Dual-acting oxazole antihypertensive agents

ABSTRACT

In one aspect, the invention relates to compounds having the formula: 
                         
wherein: Ar, Z, R 3 , R 4  and R 5  are as defined in the specification, or a pharmaceutically acceptable salt thereof. These compounds have AT 1  receptor antagonist activity and neprilysin inhibition activity. In another aspect, the invention relates to pharmaceutical compositions comprising such compounds; methods of using such compounds; and process and intermediates for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/682,299, nowallowed; which is a divisional of U.S. Ser. No. 12/835,999, now U.S.Pat. No. 8,372,984; which claims the benefit of U.S. ProvisionalApplication No. 61/227,473, filed on Jul. 22, 2009; the entiredisclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel compounds having angiotensin IItype 1 (AT₁) receptor antagonist activity and neprilysin-inhibitionactivity. The invention also relates to pharmaceutical compositionscomprising such compounds, processes and intermediates for preparingsuch compounds and methods of using such compounds to treat diseasessuch as hypertension.

2. State of the Art

The aim of antihypertensive therapy is to lower blood pressure andprevent hypertension-related complications such as myocardialinfarction, stroke, and renal disease. For patients with uncomplicatedhypertension (that is, no risk factors, target organ damage, orcardiovascular disease), it is hoped that reducing blood pressure willprevent development of cardiovascular and renal comorbidities,conditions that exist at the same time as the primary condition in thesame patient. For those patients with existing risk factors orcomorbidities, the therapeutic target is the slowing of comorbid diseaseprogression and reduced mortality.

Physicians generally prescribe pharmacological therapies for patientswhose blood pressure cannot be adequately controlled by dietary and/orlifestyle modifications. Commonly used therapeutic classes act topromote diuresis, adrenergic inhibition, or vasodilation. A combinationof drugs is often prescribed, depending upon what comorbidities arepresent.

There are five common drug classes used to treat hypertension:diuretics, which include thiazide and thiazide-like diuretics such ashydrochlorothiazide, loop diuretics such as furosemide, andpotassium-sparing diuretics such as triamterene; β₁ adrenergic receptorblockers such as metoprolol succinate and carvedilol; calcium channelblockers such as amlodipine; angiotensin-converting enzyme (ACE)inhibitors such as captopril, benazepril, enalapril, enalaprilat,lisinopril, quinapril, and ramipril; and AT₁ receptor antagonists, alsoknown as angiotensin II type 1 receptor blockers (ARBs), such ascandesartan cilexetil, eprosartan, irbesartan, losartan, olmesartanmedoxomil, telmisartan, and valsartan. Combinations of these drugs arealso administered, for example, a calcium channel blocker (amlodipine)and an ACE inhibitor (benazepril), or a diuretic (hydrochlorothiazide)and an ACE inhibitor (enalapril). All of these drugs, when usedappropriately, are effective in the treatment of hypertension.Nevertheless, both efficacy and tolerability should be further improvedin new drugs targeting hypertension. Despite the availability of manytreatment options, the recent National Health And Nutrition ExaminationSurvey (NHANES) demonstrated that only about 50% of all treated patientswith hypertension achieve adequate blood pressure control. Furthermore,poor patient compliance due to tolerability issues with availabletreatments further reduces treatment success.

In addition, each of the major classes of antihypertensive agents havesome drawbacks. Diuretics can adversely affect lipid and glucosemetabolism, and are associated with other side effects, includingorthostatic hypotension, hypokalemia, and hyperuricemia. Beta blockerscan cause fatigue, insomnia, and impotence; and some beta blockers canalso cause reduced cardiac output and bradycardia, which may beundesirable in some patient groups. Calcium channel blockers are widelyused but it is debatable as to how effectively these drugs reduce fataland nonfatal cardiac events relative to other drug classes. ACEinhibitors can cause coughing, and rarer side effects include rash,angioedema, hyperkalemia, and functional renal failure. AT₁ receptorantagonists are equally effective as ACE inhibitors but without the highprevalence of cough.

Neprilysin (neutral endopeptidase, EC 3.4.24.11) (NEP), is anendothelial membrane bound Zn²⁺ metallopeptidase found in many tissues,including the brain, kidney, lungs, gastrointestinal tract, heart, andperipheral vasculature. NEP is responsible for the degradation andinactivation of a number of vasoactive peptides, such as circulatingbradykinin and angiotensin peptides, as well as the natriureticpeptides, the latter of which have several effects includingvasodilation and diuresis. Thus, NEP plays an important role in bloodpressure homeostasis. NEP inhibitors have been studied as potentialtherapeutics, and include thiorphan, candoxatril, and candoxatrilat. Inaddition, compounds have also been designed that inhibit both NEP andACE, and include this class of compounds are described in Robl et al.(1999) Exp. Opin. Ther. Patents 9(12): 1665-1677.

There may be an opportunity to increase anti-hypertensive efficacy whencombining AT₁ receptor antagonism and NEP inhibition, as evidenced byAT₁ receptor antagonist/NEP inhibitor combinations described in WO9213564 to Darrow et al (Schering Corporation); US20030144215 to Ksanderet al.; Pu et al., Abstract presented at the Canadian CardiovascularCongress (October 2004); and Gardiner et al. (2006) JPET 319:340-348;and WO 2007/045663 (Novartis AG) to Glasspool et al.

Recently, WO 2007/056546 (Novartis AG) to Feng et al. has describedcomplexes of an AT₁ receptor antagonist and a NEP inhibitor, where anAT₁ receptor antagonist compound is non-covalently bound to a NEPinhibitor compound, or where the antagonist compound is linked vianon-covalent bonding to the inhibitor compound.

In spite of the advances in the art, there remains a need for a highlyefficacious monotherapy with multiple mechanisms of action leading tolevels of blood pressure control that can currently only be achievedwith combination therapy. Thus, although various hypertensive agents areknown, and administered in various combinations, it would be highlydesirable to provide compounds having both AT₁ receptor antagonistactivity and NEP inhibition activity in the same molecule. Compoundspossessing both of these activities are expected to be particularlyuseful as therapeutic agents since they would exhibit antihypertensiveactivity through two independent modes of action while having singlemolecule pharmacokinetics.

In addition, such dual-acting compounds are also expected to haveutility to treat a variety of other diseases that can be treated byantagonizing the AT₁ receptor and/or inhibiting the NEP enzyme.

SUMMARY OF THE INVENTION

The present invention provides novel compounds that have been found topossess AT₁ receptor antagonist activity and neprilysin (NEP) enzymeinhibition activity. Accordingly, compounds of the invention areexpected to be useful and advantageous as therapeutic agents fortreating conditions such as hypertension and heart failure.

One aspect of the invention relates to a compound of formula I:

where: Z is an oxazole selected from:

Ar is selected from:

R¹ is selected from —COOR, —SO₂NHC(O)R^(1a), tetrazolyl,

where R^(1a) is —C₁₋₆alkyl, —C₀₋₆alkylene-OR, —C₃₋₇cycloalkyl,—C₀₋₅alkylene-NRR, pyridyl, isoxazolyl, methylisoxazolyl, pyrrolidinyl,morpholinyl, and phenyl optionally substituted with halo; where each Ris independently selected from H and —C₁₋₆alkyl;

a is 0, 1, or 2; R² is F;

R³ is selected from —C₂₋₅alkyl and —O—C₁₋₅alkyl;

R⁴ is selected from —CH₂—SR^(4a), —CH₂—N(OH)C(O)H,—CH(R^(4b))C(O)NH(OH), and —CH(R^(4b))COOR^(4c); where R^(4a) is H or—C(O)—C₁₋₆alkyl; R^(4b) is H or —OH; and R^(4a) is H or —C₁₋₆alkyl; and

R⁵ is selected from —C₁₋₆alkyl, —CH₂-furanyl, —CH₂-thiophenyl, benzyl,and benzyl substituted with one or more halo, —CH₃, or —CF₃ groups;

where each ring in Ar is optionally substituted with 1 to 3 substituentsindependently selected from —OH, —C₁₋₆alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl,—CN, halo, —O—C₁₋₆alkyl, —S—C₁₋₆alkyl, —S(O)₂—C₁₋₄alkyl, -phenyl, —NO₂,—NH₂, —NH—C₁₋₆alkyl and —N(C₁₋₆alkyO₂, wherein each alkyl, alkenyl andalkynyl is optionally substituted with 1 to 5 fluoro atoms;

or a pharmaceutically acceptable salt thereof.

Another aspect of the invention relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a compound of theinvention. Such compositions may optionally contain other therapeuticagents such as diuretics, β₁ adrenergic receptor blockers, calciumchannel blockers, angiotensin-converting enzyme inhibitors, AT₁ receptorantagonists, neprilysin inhibitors, non-steroidal anti-inflammatoryagents, prostaglandins, anti-lipid agents, anti-diabetic agents,anti-thrombotic agents, renin inhibitors, endothelin receptorantagonists, endothelin converting enzyme inhibitors, aldosteroneantagonists, angiotensin-converting enzyme/neprilysin inhibitors,vasopressin receptor antagonists, and combinations thereof. Accordingly,in yet another aspect of the invention, a pharmaceutical compositioncomprises a compound of the invention, a second therapeutic agent, and apharmaceutically acceptable carrier. Another aspect of the inventionrelates to a combination of active agents, comprising a compound of theinvention and a second therapeutic agent. The compound of the inventioncan be formulated together or separately from the additional agent(s).When formulated separately, a pharmaceutically acceptable carrier may beincluded with the additional agent(s). Thus, yet another aspect of theinvention relates to a combination of pharmaceutical compositions, thecombination comprising: a first pharmaceutical composition comprising acompound of the invention and a first pharmaceutically acceptablecarrier; and a second pharmaceutical composition comprising a secondtherapeutic agent and a second pharmaceutically acceptable carrier. Inanother aspect, the invention relates to a kit containing suchpharmaceutical compositions, for example where the first and secondpharmaceutical compositions are separate pharmaceutical compositions.

Compounds of the invention possess both AT₁ receptor antagonist activityand NEP enzyme inhibition activity, and are therefore expected to beuseful as therapeutic agents for treating patients suffering from adisease or disorder that is treated by antagonizing the AT₁ receptorand/or inhibiting the NEP enzyme. Thus, one aspect of the inventionrelates to a method of treating patients suffering from a disease ordisorder that is treated by antagonizing the AT₁ receptor and/orinhibiting the NEP enzyme, comprising administering to a patient atherapeutically effective amount of a compound of the invention. Anotheraspect of the invention relates to a method of treating hypertension orheart failure, comprising administering to a patient a therapeuticallyeffective amount of a compound of the invention. Still another aspect ofthe invention relates to a method for antagonizing an AT₁ receptor in amammal comprising administering to the mammal, an AT₁receptor-antagonizing amount of a compound of the invention. Yet anotheraspect of the invention relates to a method for inhibiting a NEP enzymein a mammal comprising administering to the mammal, a NEPenzyme-inhibiting amount of a compound of the invention.

Compounds of the invention that are of particular interest include thosethat exhibit an inhibitory constant (pk_(i)) for binding to an AT₁receptor greater than or equal to about 5.0; in particular those havinga pK_(i) greater than or equal to about 6.0; in one embodiment thosehaving a pK_(i) greater than or equal to about 7.0; more particularlythose having a pK_(i) greater than or equal to about 8.0; and in yetanother embodiment, those having a pK_(i) within the range of about8.0-10.0. Compounds of particular interest also include those having aNEP enzyme inhibitory concentration (pIC₅₀) greater than or equal toabout 5.0; in one embodiment those having a pIC₅₀ greater than or equalto about 6.0; in particular those having a pIC₅₀ greater than or equalto about 7.0; and most particularly those having a pIC₅₀ within therange of about 7.0-10.0. Compounds of further interest include thosehaving a pK_(i) for binding to an AT₁ receptor greater than or equal toabout 7.5 and having a NEP enzyme pIC₅₀ greater than or equal to about7.0.

Since compounds of the invention possess AT₁ receptor antagonistactivity and NEP inhibition activity, such compounds are also useful asresearch tools. Accordingly, one aspect of the invention relates to amethod of using a compound of the invention as a research tool, themethod comprising conducting a biological assay using a compound of theinvention. Compounds of the invention can also be used to evaluate newchemical compounds. Thus another aspect of the invention relates to amethod of evaluating a test compound in a biological assay, comprising:(a) conducting a biological assay with a test compound to provide afirst assay value; (b) conducting the biological assay with a compoundof the invention to provide a second assay value; wherein step (a) isconducted either before, after or concurrently with step (b); and (c)comparing the first assay value from step (a) with the second assayvalue from step (b). Exemplary biological assays include an AT₁ receptorbinding assay and a NEP enzyme inhibition assay. Still another aspect ofthe invention relates to a method of studying a biological system orsample comprising an AT₁ receptor, a NEP enzyme, or both, the methodcomprising: (a) contacting the biological system or sample with acompound of the invention; and (b) determining the effects caused by thecompound on the biological system or sample.

Yet another aspect of the invention relates to processes andintermediates useful for preparing compounds of the invention.Accordingly, another aspect of the invention relates to a process ofpreparing compounds of the invention comprising the step of coupling acompound of formula 1 with a compound of formula 2:

where: Ar* represents Ar—R^(1*), where R^(1*) is R¹ or a protected formof R¹; and R^(4*) represents R⁴ or a protected form of R⁴; andoptionally deprotecting the product when R^(1*) is a protected form ofR¹ and/or R^(4*) is a protected form of R⁴. Another aspect of theinvention relates to a process of preparing a pharmaceuticallyacceptable salt of a compound of formula I, comprising contacting acompound of formula I in free acid or base form with a pharmaceuticallyacceptable base or acid. In other aspects, the invention relates toproducts prepared by any of the processes described herein, as well asnovel intermediates used in such process. In one aspect of the inventionnovel intermediates have formula V, VI or VII, as defined herein.

Yet another aspect of the invention relates to the use of a compound offormula I or a pharmaceutically acceptable salt thereof, for themanufacture of a medicament, especially for the manufacture of amedicament useful for treating hypertension or heart failure. Anotheraspect of the invention relates to use of a compound of the inventionfor antagonizing an AT₁ receptor or for inhibiting a NEP enzyme in amammal. Still another aspect of the invention relates to the use of acompound of the invention as a research tool. Other aspects andembodiments of the invention are disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention relates to compounds of formula I:

or a pharmaceutically acceptable salt thereof.

As used herein, the term “compound of the invention” includes allcompounds encompassed by formula I such as the species embodied informulas II, III, IV and V. In addition, the compounds of the inventionmay also contain several basic or acidic groups (for example, amino orcarboxyl groups) and therefore, such compounds can exist as a free base,free acid, or in various salt forms. All such salt forms are includedwithin the scope of the invention. Furthermore, the compounds of theinvention may also exist as prodrugs. Accordingly, those skilled in theart will recognize that reference to a compound herein, for example,reference to a “compound of the invention” or a “compound of formula I”includes a compound of formula I as well as pharmaceutically acceptablesalts and prodrugs of that compound unless otherwise indicated. Further,the term “or a pharmaceutically acceptable salt and/or prodrug thereof”is intended to include all permutations of salts and prodrugs, such as apharmaceutically acceptable salt of a prodrug. Furthermore, solvates ofcompounds of formula I are included within the scope of this invention.

The compounds of formula I may contain one or more chiral centers andtherefore, these compounds may be prepared and used in variousstereoisomeric forms. Accordingly, the invention relates to racemicmixtures, pure stereoisomers (enantiomers or diastereomers),stereoisomer-enriched mixtures, and the like unless otherwise indicated.When a chemical structure is depicted herein without anystereochemistry, it is understood that all possible stereoisomers areencompassed by such structure. Thus, for example, the term “compound offormula I” is intended to include all possible stereoisomers of thecompound. Similarly, when a particular stereoisomer is shown or namedherein, it will be understood by those skilled in the art that minoramounts of other stereoisomers may be present in the compositions of theinvention unless otherwise indicated, provided that the utility of thecomposition as a whole is not eliminated by the presence of such otherisomers. Individual enantiomers may be obtained by numerous methods thatare well known in the art, including chiral chromatography using asuitable chiral stationary phase or support, or by chemically convertingthem into diastereomers, separating the diastereomers by conventionalmeans such as chromatography or recrystallization, then regenerating theoriginal enantiomers. Additionally, where applicable, all cis-trans orE/Z isomers (geometric isomers), tautomeric forms and topoisomeric formsof the compounds of the invention are included within the scope of theinvention unless otherwise specified.

One possible chiral center could be present at the carbon on the —CHR⁴R⁵group, when R⁵ is a group such as —C₁₋₆alkyl, for example —CH₂CH(CH₃)₂.This chiral center is present at the carbon atom indicated by the symbol*:

In one embodiment of the invention, the carbon atom identified by thesymbol * has the (R) configuration. In this embodiment, compounds offormula I have the (R) configuration at the carbon atom identified bythe symbol * or are enriched in a stereoisomeric form having the (R)configuration at this carbon atom. In another embodiment, the carbonatom identified by the symbol * has the (S) configuration. In thisembodiment, compounds of formula I have the (S) configuration at thecarbon atom identified by the symbol * or are enriched in astereoisomeric form having the (S) configuration at this carbon atom.

The compounds of the invention can also have two chiral centers on the—CHR⁴R⁵ group, for example when R⁴ is —CH(OH)COOH and R⁵ is benzyl.These chiral centers are present at the carbons atom indicated by thesymbols * and **:

In such cases, four possible diastereomers can exist. For example, bothcarbon atoms can have the (R) configuration, and in such an embodiment,compounds of formula I have the (R) configuration at the carbon atomsidentified by the symbols * and ** or are enriched in a stereoisomericform having the (R,R) configuration at these atoms. In anotherembodiment, both carbon atoms can have the (S) configuration, and insuch an embodiment, compounds of formula I have the (S,S) configurationat the carbon atoms identified by the symbols * and ** or are enrichedin a stereoisomeric form having the (S) configuration at these atoms. Inyet another embodiment, the carbon atom identified by the symbol * canhave the (S) configuration and the carbon atom identified by the symbol** can have the (R) configuration, and in such an embodiment, compoundsof formula I have the (S,R) configuration at the carbon atoms identifiedby the symbols * and ** or are enriched in a stereoisomeric form havingthe (S,R) configuration at these atoms. In still another embodiment, thecarbon atom identified by the symbol * can have the (R) configurationand the carbon atom identified by the symbol ** can have the (S)configuration, and in such an embodiment, compounds of formula I havethe (R,S) configuration at the carbon atoms identified by the symbols *and ** or are enriched in a stereoisomeric form having the (R,S)configuration at these atoms.

In some cases, in order to optimize the therapeutic activity of thecompounds of the invention, for example, as hypertensive agents, it maybe desirable that the carbon atom identified by the symbols * and/or **have a particular (R), (S), (R,R), (S,S), (S,R), or (R,S) configuration.

The compounds of the invention, as well as those compounds used in theirsynthesis, may also include isotopically-labeled compounds, that is,where one or more atoms have been enriched with atoms having an atomicmass different from the atomic mass predominately found in nature.Examples of isotopes that may be incorporated into the compounds offormula I, for example, include, but are not limited to, ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ³⁶Cl, and ¹⁸F.

The compounds of formula I have been found to possess AT₁ receptorantagonizing activity and NEP enzyme inhibition activity. Among otherproperties, such compounds are expected to be useful as therapeuticagents for treating diseases such as hypertension. By combining dualactivity into a single compound, double therapy can be achieved, thatis, AT₁ receptor antagonist activity and NEP enzyme inhibition activitycan be obtained using a single active component. Since pharmaceuticalcompositions containing one active component are typically easier toformulate than compositions containing two active components, suchsingle-component compositions provide a significant advantage overcompositions containing two active components. In addition, certaincompounds of the invention have also been found to be selective forinhibition of the AT₁ receptor over the angiotensin II type 2 (AT₂)receptor, a property that may have therapeutic advantages.

The nomenclature used herein to name the compounds of the invention isillustrated in the Examples herein. This nomenclature has been derivedusing the commercially available AutoNom software (MDL, San Leandro,Calif.).

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of theinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of the invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from the invention unless specifically indicated.

In one aspect, the invention relates to compounds of formula I:

Z represents an oxazole selected from:

Thus, the compounds of the invention can also be depicted as formulas IIand III:

Ar represents an aryl group selected from:

The integer “a” is 0, 1, or 2, and the R² group is fluoro. Exemplaryfluoro-substituted Ar moieties include:

Each ring in Ar may also be substituted with 1 to 3 substituentsindependently selected from —OH, —C₁₋₆alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl,—CN, halo, —O—C₁₋₆alkyl, —S—C₁₋₆alkyl, —S(O)—C₁₋₆alkyl,—S(O)₂—C₁₋₄alkyl, -phenyl, —NO₂, —NH₂, —NH—C₁₋₆alkyl and —N(C₁₋₆alkyl)₂,wherein each alkyl, alkenyl and alkynyl is optionally substituted with 1to 5 fluoro atoms.

R¹ is selected from —COOR, —SO₂NHC(O)R^(1a), tetrazolyl,

The R^(1a) moiety is —C₁₋₆alkyl, —C₀₋₆alkylene-OR, —C₃₋₇cycloalkyl,—C₀₋₅alkylene-NRR, pyridyl, isoxazolyl, methylisoxazolyl, pyrrolidinyl,morpholinyl, and phenyl optionally substituted with halo. Each R isindependently selected from H and —C₁₋₆alkyl.

In one embodiment, R¹ is —COOR, for example, R¹ is —COOH or —COOCH₃.

In one particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a) is—C₁₋₆alkyl. Examples of this embodiment include —SO₂NHC(O)CH₃ andSO₂NHC(O)CH₂CH₃. In one particular embodiment, R¹ is tetrazolyl such as1H-tetrazol-5-yl or 5H-tetrazol-5-yl.

In one particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a) is—C₀₋₆alkylene-OR. Examples of this embodiment include —SO₂NHC(O)OCH₃,—SO₂NHC(O)OCH₂CH₃, —SO₂NHC(O)CH₂OCH₃, —SO₂NHC(O)CH₂OH,—SO₂NHC(O)CH(CH₃)OH, —SO₂NHC(O)C(CH₃)₂OH, —SO₂NHC(O)CH₂OCH₃, and—SO₂NHC(O)(CH₂)₂OCH₃.

In another particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a)is —C₃₋₇cycloalkyl. Examples of this embodiment include—SO₂NHC(O)-cyclopropyl. In another particular embodiment, R¹ is—SO₂NHC(O)R^(1a), where R^(1a) is —C₀₋₅alkylene-NRR. Examples of thisembodiment include SO₂NHC(O)NH(CH₃), —SO₂NHC(O)N(CH₃)₂,—SO₂NHC(O)NH(CH₂CH₃), and —SO₂NHC(O)C(CH₃)₂NH₂.

In another particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a)is pyridyl, for example, —SO₂NHC(O)-2-pyridyl, —SO₂NHC(O)-3-pyridyl, or—SO₂NHC(O)-4-pyridyl. The term “pyridyl” means a heterocyclic compoundof the formula:

which is bonded to any available point of attachment and includes:

In another particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a)is isoxazolyl, for example, —SO₂NHC(O)-3-isoxazolyl,—SO₂NHC(O)-4-isoxazolyl, and —SO₂NHC(O)-5-isoxazolyl. The term“isoxazolyl” means a heterocyclic compound of the formula:

which is bonded to any available point of attachment and includes:

In one particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a) ismethylisoxazolyl, for example —SO₂NHC(O)-3-isoxazolyl-5-methyl or—SO₂NHC(O)-5-isoxazolyl-3-methyl. The term “methylisoxazolyl” means aheterocyclic compound of the formula:

which is bonded to any available point of attachment and includes:

In another particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a)is pyrrolidinyl, for example, —SO₂NHC(O)-1-pyrrolidyl,—SO₂NHC(O)-2-pyrrolidyl, and —SO₂NHC(O)-3-pyrrolidyl. The term“pyrrolidinyl” means a heterocyclic compound of the formula:

which is bonded to any available point of attachment and includes:

In one particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a) ismorpholinyl, for example, —SO₂NHC(O)-4-morpholinyl. The term“morpholinyl” means a heterocyclic compound of the formula:

which is bonded to any available point of attachment and includes:

In yet another particular embodiment, R¹ is —SO₂NHC(O)R^(1a), whereR^(1a) is phenyl optionally substituted with halo. In one embodiment,the phenyl group is unsubstituted and R¹ is —SO₂NHC(O)phenyl. In anotherembodiment, the phenyl group is substituted with 1 or 2 halo atoms. Inyet another embodiment, the halo atoms are fluoro atoms. Examples ofthis embodiment include —SO₂NHC(O)-2-fluorophenyl.

In still another particular embodiment, R¹ is tetrazol-5-yl. In yetanother embodiment, R¹ is:

And in yet another embodiment, R¹ is:

R³ is selected from —C₂₋₅alkyl and —O—C₁₋₅alkyl. Examples of —C₂₋₅alkylinclude —CH₂CH₃, —(CH₂)₂CH₃, —CH(CH₃)₂, —(CH₂)₃CH₃, —CH₂CH(CH₃)₂,—C(CH₃)₃, CH(CH₃)—CH₂CH₃, and —(CH₂)₄CH₃. In one embodiment, R³ ispropyl, ethyl, or butyl. Examples of —O—C₁₋₅alkyl include —OCH₃,—OCH₂CH₃, —O(CH₂)₂CH₃, and —OCH(CH₃)₂. In one embodiment, R³ is ethoxy.

R⁴ is selected from —CH₂—SR^(4a), —CH₂—N(OH)C(O)H,—CH(R^(4b))C(O)NH(OH), and —CH(R^(4b))COOR^(4c). The R^(4a) moiety is Hor —C(O)—C₁₋₆alkyl. The R^(4b) moiety is H or —OH, and the R^(4c) moietyis H or —C₁₋₆alkyl.

In one particular embodiment, R⁴ is —CH₂—SR^(4a). Examples of thisembodiment include —CH₂SH and —CH₂—S—C(O)CH₃.

In another embodiment, R⁴ is —CH₂N(OH)C(O)H. In one particularembodiment, R⁴ is —CH(R^(4b))C(O)NH(OH), such as —CH₂C(O)NH(OH) or—CH(OH)C(O)NH(OH).

In one embodiment, R⁴ is —CH(R^(4b))COOR^(4c), where R^(4b) H, examplesof which include —CH₂COOH and —CH₂COOCH₃. In another embodiment, R⁴ is—CH(R^(4b))COOR^(4c), where R^(4b) is —OH, examples of which include—CH(OH)COOH and —CH(OH)COOCH₃.

In one embodiment, R⁴ is —CH(R^(4b))COOR^(4c), where R^(4b) H, and R⁵ isbenzyl substituted with one or more halo, —CH₃, or —CF₃ groups.

In one aspect, the invention relates to compounds of formula IV:

where Ar, R³, R^(4b), R^(4c), and R⁵ are as defined for formula I; or apharmaceutically acceptable salt thereof.

In one embodiment, R⁴ is selected from —CH₂—SR^(4a), —CH₂—N(OH)C(O)H,—CH(R^(4b))C(O)NH(OH), and —CH(R^(4b))COOR^(4c); where R^(4a) and R^(4c)are H; and R^(4b) is as defined for formula I. In another aspect, theseembodiments have formula II or III.

In yet another embodiment, R⁴ is selected from —CH₂—SR and—CH(R^(4b))COOR^(4c); where R^(4a) is —C(O)—C₁₋₆alkyl; R^(4c) is—C₁₋₆alkyl; and R^(4b) is as defined for formula I. In one aspect of theinvention, these compounds may find particular utility as prodrugs or asintermediates in the synthetic procedures described herein. In anotheraspect, these embodiments have formula II or III.

R⁵ is selected from —C₁₋₆alkyl, —CH₂-furanyl, —CH₂-thiophenyl, benzyl,and benzyl substituted with one or more halo, —CH₃, or —CF₃ groups. Inone particular embodiment, R⁵ is —C₁₋₆alkyl. Examples of this embodimentinclude i-butyl. In another embodiment, R⁵ is —CH₂-furanyl such as—CH₂-furan-2-yl or —CH₂-furan-3-yl. In one particular embodiment, R⁵ is—CH₂-thiophenyl such as —CH₂-thiophen-2-yl or —CH₂-thiophen-3-yl. In yetanother particular embodiment, R⁵ is benzyl. In still anotherembodiment, R⁵ is benzyl substituted with one or more halo, —CH₃, or—CF₃ groups. Examples of this embodiment include 2-bromobenzyl,2-chlorobenzyl, 2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl,2-methylbenzyl, and 2-trifluoromethylbenzyl.

In one particular embodiment, Ar is:

R¹ is —SO₂NHC(O)CH₃, —SO₂NHC(O)CH₂CH₃, —SO₂NHC(O)OCH₃,—SO₂NHC(O)OCH₂CH₃, —SO₂NHC(O)CH₂OCH₃, —SO₂NHC(O)CH₂OH,—SO₂NHC(O)CH(CH₃)OH, —SO₂NHC(O)C(CH₃)₂OH, —SO₂NHC(O)CH₂OCH₃,—SO₂NHC(O)(CH₂)₂OCH₃, —SO₂NHC(O)-cyclopropyl, —SO₂NHC(O)NH(CH₃),—SO₂NHC(O)N(CH₃)₂, —SO₂NHC(O)NH(CH₂CH₃), —SO₂NHC(O)C(CH₃)₂NH₂,—SO₂NHC(O)-2-pyridyl, —SO₂NHC(O)-4-pyridyl, —SO₂NHC(O)-5-isoxazolyl,—SO₂NHC(O)-3-isoxazolyl-5-methyl, —SO₂NHC(O)-1-pyrrolidyl,—SO₂NHC(O)-4-morpholinyl, —SO₂NHC(O)phenyl, —SO₂NHC(O)-2-fluorophenyl,1H-tetrazol-5-yl,

R³ is propyl, ethyl, butyl, or ethoxy; R⁴ is —CH₂SH, —CH₂—S—C(O)CH₃,—CH₂N(OH)C(O)H, —CH₂C(O)NH(OH), —CH(OH)C(O)NH(OH), —CH(OH)COOH,CH(OH)COOCH₃, —CH₂COOH, or —CH₂COOCH₃; R⁵ is i-butyl, —CH₂-furan-2-yl,—CH₂-thiophen-3-yl, benzyl, 2-bromobenzyl, 2-chlorobenzyl,2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl, 2-methylbenzyl, or2-trifluoromethylbenzyl; or a pharmaceutically acceptable salt thereof.In another aspect, this embodiment has formula II or III. In anotheraspect, when R⁴ is —CH(OH)COOH, CH(OH)COOCH₃, —CH₂COOH, or —CH₂COOCH₃,this embodiment has formula IV.

In addition, particular compounds of formula I that are of interestinclude those set forth in the Examples below, as well apharmaceutically acceptable salt thereof.

DEFINITIONS

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated. Additionally, as used herein, the singular forms“a,” “an,” and “the” include the corresponding plural forms unless thecontext of use clearly dictates otherwise. The terms “comprising”,“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements. Allnumbers expressing quantities of ingredients, properties such asmolecular weight, reaction conditions, and so forth used herein are tobe understood as being modified in all instances by the term “about,”unless otherwise indicated. Accordingly, the numbers set forth hereinare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each number should at least be construed in lightof the reported significant digits and by applying ordinary roundingtechniques.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms and include, for example,—C₁₋₄alkyl, —C₁₋₅alkyl, —C₂₋₅alkyl, —C₁₋₆alkyl, and —C₁₋₁₀alkyl.Representative alkyl groups include, by way of example, methyl, ethyl,n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, n-pentyl,n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown preceding the term assubscript. For example, the term “—C₁₋₆alkyl” means an alkyl grouphaving from 1 to 6 carbon atoms, and the term “—C₃₋₇cycloalkyl” means acycloalkyl group having from 3 to 7 carbon atoms, respectively, wherethe carbon atoms are in any acceptable configuration.

The term “alkylene” means a divalent saturated hydrocarbon group thatmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 0 to 10 carbon atoms and include, forexample, —C₀₋₁alkylene-, —C₀₋₂alkylene-, —C₀₋₃alkylene-, —C₀₋₅alkylene-,—C₀₋₆alkylene-, —C₁₋₂alkylene- and —C₁₋₁₂alkylene-. Representativealkylene groups include, by way of example, methylene, ethane-1,2-diyl(“ethylene”), propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl,pentane-1,5-diyl and the like. It is understood that when the alkyleneterm include zero carbons such as —C₀₋₅alkylene- or —C₀₋₆alkylene-, suchterms are intended to include the absence of carbon atoms, that is, thealkylene group is not present except for a covalent bond attaching thegroups separated by the alkylene term.

The term “alkoxy” means a monovalent group of the formula —O-alkyl,where alkyl is as defined herein. Unless otherwise defined, such alkoxygroups typically contain from 1 to 10 carbon atoms and include, forexample, —O—C₁₋₄alkyl and —O—C₁₋₅alkyl. Representative alkoxy groupsinclude, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy,n-butoxy, sec-butoxy, isobutoxy, t-butoxy and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms and include, for example,—C₃₋₅cycloalkyl, —C₃₋₆cycloalkyl and —C₃₋₇cycloalkyl. Representativecycloalkyl groups include, by way of example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and the like. The term “cycloalkylene” means adivalent aryl group such as —C₄₋₈cycloalkylene.

The term “halo” means fluoro, chloro, bromo and iodo.

As used herein, the phrase “having the formula” or “having thestructure” is not intended to be limiting and is used in the same waythat the term “comprising” is commonly used.

The term “optionally substituted” means that group in question may beunsubstituted or it may be substituted one or several times, such as 1to 3 times or 1 to 5 times. For example, a phenyl group that is“optionally substituted” with halo atoms, may be unsubstituted, or itmay contain 1, 2, 3, 4, or 5 halo atoms.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise unacceptable when used in the invention. Forexample, the term “pharmaceutically acceptable carrier” refers to amaterial that can be incorporated into a composition and administered toa patient without causing unacceptable biological effects or interactingin an unacceptable manner with other components of the composition. Suchpharmaceutically acceptable materials typically have met the requiredstandards of toxicological and manufacturing testing, and include thosematerials identified as suitable inactive ingredients by the U.S. Foodand Drug administration.

The term “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (for example, salts having acceptable mammalian safetyfor a given dosage regime). However, it is understood that the saltscovered by the invention are not required to be pharmaceuticallyacceptable salts, such as salts of intermediate compounds that are notintended for administration to a patient. Pharmaceutically acceptablesalts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. In addition, when a compound of formula I contains both a basicmoiety, such as an amine, pyridine or imidazole, and an acidic moietysuch as a carboxylic acid or tetrazole, zwitterions may be formed andare included within the term “salt” as used herein. Salts derived frompharmaceutically acceptable inorganic bases include ammonium, calcium,copper, ferric, ferrous, lithium, magnesium, manganic, manganous,potassium, sodium, and zinc salts, and the like. Salts derived frompharmaceutically acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable inorganic acids include salts of boric,carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric orhydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Saltsderived from pharmaceutically acceptable organic acids include salts ofaliphatic hydroxyl acids (for example, citric, gluconic, glycolic,lactic, lactobionic, malic, and tartaric acids), aliphaticmonocarboxylic acids (for example, acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (for example, aspartic andglutamic acids), aromatic carboxylic acids (for example, benzoic,p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylaceticacids), aromatic hydroxyl acids (for example, o-hydroxybenzoic,p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic and3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic acids(for example, fumaric, maleic, oxalic and succinic acids), glucoronic,mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids(for example, benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic,isethionic, methanesulfonic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic andp-toluenesulfonic acids), xinafoic acid, and the like.

The term “protected derivatives thereof” means a derivative of thespecified compound in which one or more functional groups of thecompound are protected or blocked from undergoing undesired reactionswith a protecting or blocking group. Functional groups that may beprotected include, by way of example, carboxy groups, amino groups,hydroxyl groups, thiol groups, carbonyl groups and the like.Representative protecting groups for carboxy groups include esters (suchas a p-methoxybenzyl ester), amides and hydrazides; for amino groups,carbamates (such as t-butoxycarbonyl) and amides; for hydroxyl groups,ethers and esters; for thiol groups, thioethers and thioesters; forcarbonyl groups, acetals and ketals; and the like. Such protectinggroups are well known to those skilled in the art and are described, forexample, in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Third Edition, Wiley, New York, 1999, and references citedtherein.

As used herein, the term “prodrug” is intended to mean an inactive (orsignificantly less active) precursor of a drug that is converted intoits active form in the body under physiological conditions, for example,by normal metabolic processes. The term is also intended to includecertain protected derivatives of compounds of formula I that may be madeprior to a final deprotection stage. Such compounds may not possesspharmacological activity at AT₁ and/or NEP, but may be administeredorally or parenterally and thereafter metabolized in the body to formcompounds of the invention which are pharmacologically active at AT₁and/or NEP. Thus, all protected derivatives and prodrugs of compoundsformula I are included within the scope of the invention. Prodrugs ofcompounds of formula I having a free carboxyl, sulfhydryl or hydroxygroup can be readily synthesized by techniques that are well known inthe art. These prodrug derivatives are then converted by solvolysis orunder physiological conditions to be the free carboxyl, sulfhydryland/or hydroxy compounds. Exemplary prodrugs include: esters includingC₁₋₆alkylesters and aryl-C₁₋₆alkylesters, carbonate esters, hemi-esters,phosphate esters, nitro esters, sulfate esters, sulfoxides, amides,carbamates, azo-compounds, phosphamides, glycosides, ethers, acetals,ketals, and disulfides. In one embodiment, the compounds of formula Ihave a free sulfhydryl or a free carboxyl and the prodrug is an esterderivative thereof, i.e., the prodrug is a thioester such as —SC(O)CH₃or an ester such as —C(O)OCH₃.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, for example, a compound of formula I or apharmaceutically acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include, by way of example, water, methanol, ethanol,isopropanol, acetic acid and the like. When the solvent is water, thesolvate formed is a hydrate.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need thereof, thatis, the amount of drug needed to obtain the desired therapeutic effect.For example, a therapeutically effective amount for treatinghypertension is an amount of compound needed to, for example, reduce,suppress, eliminate or prevent the symptoms of hypertension, or to treatthe underlying cause of hypertension. In one embodiment, atherapeutically effective amount is that amount of drug needed to reduceblood pressure or the amount of drug needed to maintain normal bloodpressure. On the other hand, the term “effective amount” means an amountsufficient to obtain a desired result, which may not necessary be atherapeutic result. For example, when studying a system comprising anAT₁ receptor, an “effective amount” may be the amount needed toantagonize the receptor.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as hypertension) in apatient, such as a mammal (particularly a human) that includes one ormore of the following: (a) preventing the disease or medical conditionfrom occurring, that is, by prophylactic treatment of a patient; (b)ameliorating the disease or medical condition such as by eliminating orcausing regression of the disease or medical condition in a patient; (c)suppressing the disease or medical condition such as by slowing orarresting the development of the disease or medical condition in apatient; or (d) alleviating the symptoms of the disease or medicalcondition in a patient. For example, the term “treating hypertension”would include preventing hypertension from occurring, amelioratinghypertension, suppressing hypertension, and alleviating the symptoms ofhypertension (for example, lowering blood pressure). The term “patient”is intended to include those mammals, such as humans, that are in needof treatment or disease prevention or that are presently being treatedfor disease prevention or treatment of a specific disease or medicalcondition, as well as test subjects in which compounds of the inventionare being evaluated or being used in a assay, for example an animalmodel.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods, the proceduresset forth in the Examples, or by using other methods, reagents, andstarting materials that are known to those of ordinary skill in the art.Although the following procedures may illustrate a particular embodimentof the invention, it is understood that other embodiments of theinvention can be similarly prepared using the same or similar methods orby using other methods, reagents and starting materials known to thoseof ordinary skill in the art. It will also be appreciated that wheretypical or preferred process conditions (for example, reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. While optimum reaction conditions will typically varydepending on various reaction parameters such as the particularreactants, solvents and quantities used, those of ordinary skill in theart can readily determine suitable reaction conditions using routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions and reagents for protection anddeprotection of such functional groups are well-known in the art.Protecting groups other than those illustrated in the proceduresdescribed herein may be used, if desired. For example, numerousprotecting groups, and their introduction and removal, are described inT. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis,Third Edition, Wiley, New York, 1999, and references cited therein. Morespecifically, the following abbreviations and reagents are used in theschemes presented below:

P¹ represents an “amino-protecting group,” a term used herein to mean aprotecting group suitable for preventing undesired reactions at an aminogroup. Representative amino-protecting groups include, but are notlimited to, t-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl(Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl (TMS),t-butyldimethylsilyl (TBDMS), and the like. Standard deprotectiontechniques are used to remove the P¹ group. For example, a BOC group canbe removed using an acidic reagent such as TFA in DCM or HCl in1,4-dioxane, while a Cbz group can be removed by employing catalytichydrogenation conditions such as H₂ (1 atm) and 10% Pd/C in an alcoholicsolvent (“H₂/Pd/C”).

P² represents a “carboxy-protecting group,” a term used herein to mean aprotecting group suitable for preventing undesired reactions at acarboxy group. Representative carboxy-protecting groups include, but arenot limited to, methyl, ethyl, t-butyl, benzyl (Bn), p-methoxybenzyl(PMB), 9-fluorenylmethyl (Fm), trimethylsilyl (TMS),t-butyldimethylsilyl (TBDMS), diphenylmethyl (benzhydryl, DPM) and thelike. Standard deprotection techniques and reagents are used to removethe P² group, and may vary depending upon which group is used. Forexample, sodium or lithium hydroxide is commonly used when P² is methyl,an acid such as TFA or HCl is commonly used when P² is t-butyl, andH₂/Pd/C may be used when P² is benzyl.

P³ represents a “thiol-protecting group,” a term used herein to mean aprotecting group suitable for preventing undesired reactions at a thiolgroup. Representative thiol-protecting groups include, but are notlimited to, ethers, esters such as —C(O)CH₃, and the like. Standarddeprotection techniques and reagents such as NaOH, primary alkylamines,and hydrazine, may be used to remove the P³ group.

P⁴ represents a “tetrazole-protecting group,” a term used herein to meana protecting group suitable for preventing undesired reactions at atetrazole group. Representative tetrazole-protecting groups include, butare not limited to trityl and diphenylmethyl. Standard deprotectiontechniques and reagents such as TFA in DCM or HCl in 1,4-dioxane may beused to remove the P⁴ group.

P⁵ represents a “hydroxyl-protecting group,” a term that is used hereinto mean a protecting group suitable for preventing undesired reactionsat a hydroxyl group. Representative hydroxyl-protecting groups include,but are not limited to C₁₋₆alkyls, silyl groups includingtriC₁₋₆alkylsilyl groups, such as trimethylsilyl (TMS), triethylsilyl(TES), and tert-butyldimethylsilyl (TBDMS); esters (acyl groups)including C₁₋₆alkanoyl groups, such as formyl, acetyl, and pivaloyl, andaromatic acyl groups such as benzoyl; arylmethyl groups such as benzyl(Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl(benzhydryl, DPM); and the like. Standard deprotection techniques andreagents are used to remove the P⁵ group, and may vary depending uponwhich group is used. For example, H₂/Pd/C is commonly used when P⁵ isbenzyl, while NaOH is commonly used when P⁵ is an acyl group.

P⁶ represents a “sulfonamide-protecting group,” a term that is usedherein to mean a protecting group suitable for preventing undesiredreactions at a sulfonamide group. Representative sulfonamide-protectinggroups include, but are not limited to t-butyl and acyl groups.Exemplary acyl groups include aliphatic lower acyl groups such as theformyl, acetyl, phenylacetyl, butyryl, isobutyryl, valeryl, isovaleryland pivaloyl groups, and aromatic acyl groups such as the benzoyl and4-acetoxybenzoyl. Standard deprotection techniques and reagents are usedto remove the P⁶ group, and may vary depending upon which group is used.For example, HCl is commonly used when P⁶ is t-butyl, while NaOH iscommonly used when P⁶ is an acyl group.

In addition, L is used to designate a “leaving group,” a term usedherein to mean a functional group or atom which can be displaced byanother functional group or atom in a substitution reaction, such as anucleophilic substitution reaction. By way of example, representativeleaving groups include chloro, bromo and iodo groups; sulfonic estergroups, such as mesylate, triflate, tosylate, brosylate, nosylate andthe like; and acyloxy groups, such as acetoxy, trifluoroacetoxy and thelike.

Suitable bases for use in these schemes include, by way of illustrationand not limitation, potassium carbonate, calcium carbonate, sodiumcarbonate, triethylamine, pyridine, 1,8-diazabicyclo-[5.4.0]undec-7-ene(DBU), N,N-diisopropylethylamine (DIPEA), sodium hydroxide, potassiumhydroxide, potassium t-butoxide, and metal hydrides.

Suitable inert diluents or solvents for use in these schemes include, byway of illustration and not limitation, tetrahydrofuran (THF),acetonitrile (MeCN), N,N-dimethylformamide (DMF), dimethyl sulfoxide(DMSO), toluene, dichloromethane (DCM), chloroform (CHCl₃), carbontetrachloride (CCl₄), 1,4-dioxane, methanol, ethanol, water, and thelike.

Suitable carboxylic acid/amine coupling reagents includebenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), O-(7-azabenzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU), dicyclohexylcarbodiimide (DCC),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC),carbonyldiimidazole (CDI), and the like. Coupling reactions areconducted in an inert diluent in the presence of a base such as DIPEA,and are performed under conventional amide bond-forming conditions.

All reactions are typically conducted at a temperature within the rangeof about −78° C. to 100° C., for example at room temperature. Reactionsmay be monitored by use of thin layer chromatography (TLC), highperformance liquid chromatography (HPLC), and/or LCMS until completion.Reactions may be complete in minutes, or may take hours, typically from1-2 hours and up to 48 hours. Upon completion, the resulting mixture orreaction product may be further treated in order to obtain the desiredproduct. For example, the resulting mixture or reaction product may besubjected to one or more of the following procedures: concentrating orpartitioning (for example, between EtOAc and water or between 5% THF inEtOAc and 1M phosphoric acid); extraction (for example, with EtOAc,CHCl₃, DCM, chloroform); washing (for example, with saturated aqueousNaCl, saturated NaHCO₃, Na₂CO₃ (5%), CHCl₃ or 1M NaOH); drying (forexample, over MgSO₄, over Na₂SO₄, or in vacuo); filtering; crystallizing(for example, from EtOAc and hexane); being concentrated (for example,in vacuo); and/or purification (e.g., silica gel chromatography, flashchromatography, preparative HPLC, reverse phase-HPLC, orcrystallization).

By way of illustration, compounds of formula I, as well as their salts,solvates, and prodrugs can be prepared by coupling a compound of formula1 with a compound of formula 2:

Ar* represents Ar—R^(1*), where R^(1*) is R¹ or a protected form of R¹,for example, -tetrazolyl-BOC or a precursor of R¹ such as —CN that isthen converted to tetrazolyl. R^(4*) represents R⁴ or a protected formof R⁴. Therefore, when R^(1*) represents R¹ and R^(4*) represents R⁴,the reaction is complete after the coupling step.

On the other hand, when R^(1*) represents a protected form of R¹ and/orR^(4*) represents a protected form of R⁴, a subsequent global orsequential deprotection step would yield the non-protected compound.Similarly, when R^(1*) represents a precursor of a subsequent conversionstep would yield the desired compound. Reagents and conditions for thedeprotection vary with the nature of protecting groups in the compound.Thus, one method of preparing compounds of the invention involvescoupling compounds (1) and (2), with an optional deprotection step whenR^(1*) is a protected form of R¹ and/or R^(4*) is a protected form ofR⁴, thus forming a compound of formula I or a pharmaceuticallyacceptable salt thereof.

Examples of compound of formula 1 include:

-   5-propyl-4-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]oxazole-2-carboxylic    acid;-   5-ethoxy-4-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]oxazole-2-carboxylic    acid;-   4-[2-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5-propyloxazole-2-carboxylic    acid;-   4-[2-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5-ethoxyoxazole-2-carboxylic    acid;-   4-[3-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5-propyloxazole-2-carboxylic    acid;-   4-[3-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5-ethoxyoxazole-2-carboxylic    acid;-   4-[3,5-difluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5-propyloxazole-2-carboxylic    acid;-   4-[3,5-difluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5-ethoxyoxazole-2-carboxylic    acid;-   4-propyl-5-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]oxazole-2-carboxylic    acid;-   4-ethoxy-5-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]oxazole-2-carboxylic    acid;-   5-[2-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-4-propyloxazole-2-carboxylic    acid;-   5-[2-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-4-ethoxyoxazole-2-carboxylic    acid;-   5-[3-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-4-propyloxazole-2-carboxylic    acid;-   5-[3-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-4-ethoxyoxazole-2-carboxylic    acid;-   5-[3,5-difluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-4-propyloxazole-2-carboxylic    acid;-   5-[3,5-difluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-4-ethoxyoxazole-2-carboxylic    acid;-   4-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)-5-propyloxazole-2-carboxylic    acid;-   4-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)-5-ethoxyoxazole-2-carboxylic    acid;-   4-[2-fluoro-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)]-5-propyloxazole-2-carboxylic    acid;-   4-[2-fluoro-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)]-5-ethoxyoxazole-2-carboxylic    acid;-   4-[3-fluoro-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)]-5-propyloxazole-2-carboxylic    acid;-   4-[3-fluoro-(2′-ACetylsulfamoylbiphenyl-4-ylmethyl)]-5-ethoxyoxazole-2-carboxylic    acid;-   4-[3,5-difluoro-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)]-5-propyloxazole-2-carboxylic    acid;-   4-[3,5-difluoro-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)]-5-ethoxyoxazole-2-carboxylic    acid;-   5-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)-4-propyloxazole-2-carboxylic    acid;-   5-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)-4-ethoxyoxazole-2-carboxylic    acid;-   5-[2-fluoro-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)]-4-propyloxazole-2-carboxylic    acid;-   5-[2-fluoro-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)]-4-ethoxyoxazole-2-carboxylic    acid;-   5-[3-fluoro-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)]-4-propyloxazole-2-carboxylic    acid;-   5-[3-fluoro-2(2′-acetylsulfamoylbiphenyl-4-ylmethyl)]-4-ethoxyoxazole-2-carboxylic    acid;-   5-[3,5-difluoro-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)]-4-propyloxazole-2-carboxylic    acid; and-   5-[3,5-difluoro-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)]-4-ethoxyoxazole-2-carboxylic    acid.

Examples of compounds of formula 2 include:

-   (R)-2-((R)-2-amino-3-phenylpropyldisulfanyl)-1-benzylethylamine;-   (R)-1-((R)-2-amino-4-methylpentyldisulfanylmethyl)-3-methylbutylamine    (dimer);-   (R)-3-amino-4-(2-chlorophenyl)butyric acid;-   (R)-3-amino-4-(2-bromophenyl)butyric acid;-   (R)-3-amino-4-phenylbutyric acid;-   (R)-3-amino-4-(2-fluorophenyl)butyric acid;-   (R)-3-amino-4-(2-methylphenyl)butyric acid;-   (R)-3-amino-4-(2-trifluoromethylphenyl)butyric acid;-   (2R,3R)-3-amino-2-hydroxy-4-phenylbutyric acid ethyl ester;-   3-amino-4-(2-chlorophenyl)-2-hydroxybutyric acid ethyl ester;-   [(S)-1-benzyl-2-(benzyloxyformylamino)ethyl]carbamic acid t-butyl    ester;-   [1-benzyl-2-hydroxycarbamoylethyl]carbamic acid t-butyl ester;-   [1-(2-chlorobenzyl)-2-hydroxycarbamoylethyl]carbamic acid t-butyl    ester; and-   3-amino-2,N-dihydroxy-4-phenylbutyramide.    Other protected forms, deprotected forms, isomers and racemic    mixtures can also be used.

Certain intermediates described herein are believed to be novel andaccordingly, such compounds are provided as further aspects of theinvention including, for example, the compounds of formulas V, VI andVII, or a salt thereof:

where Ar* is Ar—R^(1*); Ar, Z, R³, R⁴, and R⁵ are as defined for formulaI; and R^(1*) is —SO₂NH—P⁶ or tetrazolyl-P⁴; where P⁴ is atetrazole-protecting group and P⁶ is a sulfonamide-protecting group;

where Ar, Z, R³, and R⁵ are as defined for formula I; R^(4*) is—CH₂—S—P³, —CH₂—N(O—P⁵)—C(O)H, —CH(R^(4b))C(O)NH(O—P⁵), or—CH(R^(4b))COO—P²; and R^(4b) is as defined for formula I; where P² is acarboxy-protecting group, P³ is a thiol-protecting group, P⁵ is ahydroxyl-protecting group; and

where Ar* is Ar—R^(1*); Ar, Z, R³, and R⁵ are as defined for formula I;R^(1*) is —SO₂NH—P⁶ or tetrazolyl-P⁴; R^(4*) is —CH₂—S—P³,—CH₂—N(O—P⁵)—C(O)H, —CH(R^(4b))C(O)NH(O—P⁵), or —CH(R^(4b))COO—P²; andR^(4b) is as defined for formula I; where P² is a carboxy-protectinggroup, P³ is a thiol-protecting group, P⁴ is a tetrazole-protectinggroup, P⁵ is a hydroxyl-protecting group, and P⁶ is asulfonamide-protecting group. Thus, another method of preparingcompounds of the invention involves deprotecting a compound of formulaV, VI, or VII.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereof are described in the Examples set forth below.

UTILITY

Compounds of the invention possess angiotensin II type 1 (AT₁) receptorantagonist activity. In one embodiment, compounds of the invention areselective for inhibition of the AT₁ receptor over the AT₂ receptor.Compounds of the invention also possess neprilysin (NEP) inhibitionactivity, that is, the compounds are able to inhibit enzyme-substrateactivity. In another embodiment, the compounds do not exhibitsignificant inhibitory activity of the angiotensin-converting enzyme.Compounds of formula I may be active drugs as well as prodrugs. Thus,when discussing the activity of compounds of the invention, it isunderstood that any such prodrugs have the expected activity oncemetabolized.

One measure of the affinity of a compound for the AT₁ receptor is theinhibitory constant (K_(i)) for binding to the AT₁ receptor. The pK_(i)value is the negative logarithm to base 10 of the K_(i). One measure ofthe ability of a compound to inhibit NEP activity is the inhibitoryconcentration (IC₅₀), which is the concentration of compound thatresults in half-maximal inhibition of substrate conversion by the NEPenzyme. The pIC₅₀ value is the negative logarithm to base 10 of theIC₅₀. Compounds of the invention that have both AT₁receptor-antagonizing activity and NEP enzyme-inhibiting activity are ofparticular interest, including those that exhibit a pk_(i) at the AT₁receptor greater than or equal to about 5.0, and exhibit a pIC₅₀ for NEPgreater than or equal to about 5.0.

In one embodiment, compounds of interest have a pK_(i) at the AT₁receptor≧about 6.0, a pK_(i) at the AT₁ receptor≦about 7.0, or a pK_(i)at the AT₁ receptor≧about 8.0. Compounds of interest also include thosehaving a pIC₅₀ for NEP≧about 6.0 or a pIC₅₀ for NEP≧about 7.0. Inanother embodiment, compounds of interest have a pK_(i) at the AT₁receptor within the range of about 8.0-10.0 and a pIC₅₀ for NEP withinthe range of about 7.0-10.0.

In another embodiment, compounds of particular interest have a pK_(i)for binding to an AT₁ receptor greater than or equal to about 7.5 and aNEP enzyme pIC₅₀ greater than or equal to about 7.0. In anotherembodiment, compounds of interest have a pK_(i) greater than or equal toabout 8.0 and a pIC₅₀ greater than or equal to about 8.0.

It is noted that in some cases, compounds of the invention, while stillhaving dual activity, may possess either weak AT₁ receptor antagonistactivity or weak NEP inhibition activity. In such cases, those of skillin the art will recognize that these compounds still have utility asprimarily either a NEP inhibitor or a AT₁ receptor antagonist,respectively, or have utility as research tools.

Exemplary assays to determine properties of compounds of the invention,such as the AT₁ receptor binding and/or NEP inhibiting activity, aredescribed in the Examples and include by way of illustration and notlimitation, assays that measure AT₁ and AT₂ binding (described in Assay1), and NEP inhibition (described in Assay 2). Useful secondary assaysinclude assays to measure ACE inhibition (also described in Assay 2) andaminopeptidase P (APP) inhibition (described in Sulpizio et al. (2005)JPET 315:1306-1313). A pharmacodynamic assay to assess the in vivoinhibitory potencies for ACE, AT₁, and NEP in anesthetized rats isdescribed in Assay 3 (see also Seymour et al. (1985) Hypertension7(Suppl I):I-35-I-42 and Wigle et al. (1992) Can. J. Physiol. Pharmacol.70:1525-1528), where AT₁ inhibition is measured as the percentinhibition of the angiotensin II pressor response, ACE inhibition ismeasured as the percent inhibition of the angiotensin I pressorresponse, and NEP inhibition is measured as increased urinary cyclicguanosine 3′,5′-monophosphate (cGMP) output. Useful in vivo assaysinclude the conscious spontaneously hypertensive rat (SHR) model, whichis a renin dependent hypertension model that is useful for measuring AT₁receptor blocking (described in Assay 4; see also Intengan et al. (1999)Circulation 100(22):2267-2275 and Badyal et al. (2003) Indian Journal ofPharmacology 35:349-362), and the conscious desoxycorticosteroneacetate-salt (DOCA-salt) rat model, which is a volume dependenthypertension model that is useful for measuring NEP activity (describedin Assay 5; see also Trapani et al. (1989) J. Cardiovasc. Pharmacol.14:419-424, Intengan et al. (1999) Hypertension 34(4):907-913, andBadyal et al. (2003) supra). Both the SHR and DOCA-salt models areuseful for evaluating the ability of a test compound to reduce bloodpressure. The DOCA-salt model is also useful to measure a testcompound's ability to prevent or delay a rise in blood pressure.Compounds of the invention are expected to antagonize the AT₁ receptorand/or inhibit the NEP enzyme in any of the assays listed above, orassays of a similar nature. Thus, the aforementioned assays are usefulin determining the therapeutic utility of compounds of the invention,for example, their utility as antihypertensive agents. Other propertiesand utilities of compounds of the invention can be demonstrated usingother in vitro and in vivo assays well-known to those skilled in theart.

Compounds of the invention are expected to be useful for the treatmentand/or prevention of medical conditions responsive to AT₁ receptorantagonism and/or NEP inhibition. Thus it is expected that patientssuffering from a disease or disorder that is treated by antagonizing theAT₁ receptor and/or by inhibiting the NEP enzyme can be treated byadministering a therapeutically effective amount of a compound of theinvention. For example, by antagonizing the AT₁ receptor and thusinterfering with the action of angiotensin II on its receptors, thesecompounds are expected to find utility in preventing the increase inblood pressure produced by angiotensin II, a potent vasopressor. Inaddition, by inhibiting NEP, the compounds are also expected topotentiate the biological effects of endogenous peptides that aremetabolized by NEP, such as the natriuretic peptides, bombesin,bradykinins, calcitonin, endothelins, enkephalins, neurotensin,substance P and vasoactive intestinal peptide. For example, bypotentiating the effects of the natriuretic peptides, compounds of theinvention are expected to be useful to treat glaucoma. These compoundsare also expected to have other physiological actions, for example, onthe renal, central nervous, reproductive and gastrointestinal systems.

Compounds of the invention are expected to find utility in treatingand/or preventing medical conditions such as cardiovascular and renaldiseases. Cardiovascular diseases of particular interest include heartfailure such as congestive heart failure, acute heart failure, chronicheart failure, and acute and chronic decompensated heart failure. Renaldiseases of particular interest include diabetic nephropathy and chronickidney disease. One embodiment of the invention relates to a method fortreating hypertension, comprising administering to a patient atherapeutically effective amount of a compound of the invention.Typically, the therapeutically effective amount is the amount that issufficient to lower the patient's blood pressure. In one embodiment, thecompound is administered as an oral dosage form.

Another embodiment of the invention relates to a method for treatingheart failure, comprising administering to a patient a therapeuticallyeffective amount of a compound of the invention. Typically, thetherapeutically effective amount is the amount that is sufficient tolower blood pressure and/or improve renal functions. In one embodiment,the compound is administered as an intravenous dosage form. When used totreat heart failure, the compound may be administered in combinationwith other therapeutic agents such as diuretics, natriuretic peptides,and adenosine receptor antagonists.

Compounds of the invention are also expected to be useful inpreventative therapy, for example in preventing the progression ofcardiac insufficiency after myocardial infarction, preventing arterialrestenosis after angioplasty, preventing thickening of blood vesselwalls after vascular operations, preventing atherosclerosis, andpreventing diabetic angiopathy.

In addition, as NEP inhibitors, compounds of the invention are expectedto inhibit enkephalinase, which will inhibit the degradation ofendogenous enkephalins and thus such compounds may also find utility asanalgesics. Due to their NEP inhibition properties, compounds of theinvention are also expected to be useful as antitussive agents andantidiarrheal agents (for example, for the treatment of waterydiarrhea), as well as find utility in the treatment of menstrualdisorders, preterm labor, pre-eclampsia, endometriosis, reproductivedisorders (for example, male and female infertility, polycystic ovariansyndrome, implantation failure), and male and female sexual dysfunction,including male erectile dysfunction and female sexual arousal disorder.More specifically, the compounds of the invention are expected to beuseful in treating female sexual dysfunction, which is often defined asa female patient's difficulty or inability to find satisfaction insexual expression. This covers a variety of diverse female sexualdisorders including, by way of illustration and not limitation,hypoactive sexual desire disorder, sexual arousal disorder, orgasmicdisorder and sexual pain disorder. When used to treat such disorders,especially female sexual dysfunction, compounds of the invention may becombined with one or more of the following secondary agents: PDE5inhibitors, dopamine agonists, estrogen receptor agonists and/orantagonists, androgens, and estrogens.

The amount of the compound of the invention administered per dose or thetotal amount administered per day may be predetermined or it may bedetermined on an individual patient basis by taking into considerationnumerous factors, including the nature and severity of the patient'scondition, the condition being treated, the age, weight, and generalhealth of the patient, the tolerance of the patient to the active agent,the route of administration, pharmacological considerations such as theactivity, efficacy, pharmacokinetics and toxicology profiles of thecompound and any secondary agents being administered, and the like.Treatment of a patient suffering from a disease or medical condition(such as hypertension) can begin with a predetermined dosage or a dosagedetermined by the treating physician, and will continue for a period oftime necessary to prevent, ameliorate, suppress, or alleviate thesymptoms of the disease or medical condition. Patients undergoing suchtreatment will typically be monitored on a routine basis to determinethe effectiveness of therapy. For example, in treating hypertension,blood pressure measurements may be used to determine the effectivenessof treatment. Similar indicators for other diseases and conditionsdescribed herein, are well known and are readily available to thetreating physician. Continuous monitoring by the physician will insurethat the optimal amount of the compound of the invention will beadministered at any given time, as well as facilitating thedetermination of the duration of treatment. This is of particular valuewhen secondary agents are also being administered, as their selection,dosage, and duration of therapy may also require adjustment. In thisway, the treatment regimen and dosing schedule can be adjusted over thecourse of therapy so that the lowest amount of active agent thatexhibits the desired effectiveness is administered and, further, thatadministration is continued only so long as is necessary to successfullytreat the disease or medical condition.

Since compounds of the invention possess AT₁ receptor antagonistactivity and/or NEP enzyme inhibition activity, such compounds are alsouseful as research tools for investigating or studying biologicalsystems or samples having AT₁ receptors or a NEP enzyme, for example tostudy diseases where the AT₁ receptor or NEP enzyme plays a role. Anysuitable biological system or sample having AT₁ receptors and/or a NEPenzyme may be employed in such studies which may be conducted either invitro or in vivo. Representative biological systems or samples suitablefor such studies include, but are not limited to, cells, cellularextracts, plasma membranes, tissue samples, isolated organs, mammals(such as mice, rats, guinea pigs, rabbits, dogs, pigs, humans, and soforth), and the like, with mammals being of particular interest. In oneparticular embodiment of the invention an AT₁ receptor in a mammal isantagonized by administering an AT₁-antagonizing amount of a compound ofthe invention. In another particular embodiment, NEP enzyme activity ina mammal is inhibited by administering a NEP-inhibiting amount of acompound of the invention. Compounds of the invention can also be usedas research tools by conducting biological assays using such compounds.

When used as a research tool, a biological system or sample comprisingan AT₁ receptor and/or a NEP enzyme is typically contacted with an AT₁receptor-antagonizing or NEP enzyme-inhibiting amount of a compound ofthe invention. After the biological system or sample is exposed to thecompound, the effects of antagonizing the AT₁ receptor and/or inhibitingthe NEP enzyme are determined using conventional procedures andequipment, such as by measuring receptor binding in a binding assay ormeasuring ligand-mediated changes in a functional assay. Exposureencompasses contacting cells or tissue with the compound, administeringthe compound to a mammal, for example by i.p., i.v. or s.c.administration, and so forth. This determining step can involvemeasuring a response (a quantitative analysis) or can involve making anobservation (a qualitative analysis). Measuring a response involves, forexample, determining the effects of the compound on the biologicalsystem or sample using conventional procedures and equipment, such asradioligand binding assays and measuring ligand-mediated changes infunctional assays. The assay results can be used to determine theactivity level as well as the amount of compound necessary to achievethe desired result, that is, an AT₁ receptor-antagonizing and/or a NEPenzyme-inhibiting amount. Typically, the determining step will involvedetermining the AT₁ receptor ligand-mediated effects and/or determiningthe effects of inhibiting the NEP enzyme.

Additionally, compounds of the invention can be used as research toolsfor evaluating other chemical compounds, and thus are also useful inscreening assays to discover, for example, new compounds having AT₁receptor-antagonizing activity and/or NEP-inhibiting activity. In thismanner, a compound of the invention is used as a standard in an assay toallow comparison of the results obtained with a test compound and withcompounds of the invention to identify those test compounds that haveabout equal or superior activity, if any. For example, K_(i) data (asdetermined, for example, by a binding assay) for a test compound or agroup of test compounds is compared to the K_(i) data for a compound ofthe invention to identify those test compounds that have the desiredproperties, for example, test compounds having a K_(i) value about equalor superior to a compound of the invention, if any. This aspect of theinvention includes, as separate embodiments, both the generation ofcomparison data (using the appropriate assays) and the analysis of testdata to identify test compounds of interest. Thus, a test compound canbe evaluated in a biological assay, by a method comprising the steps of:(a) conducting a biological assay with a test compound to provide afirst assay value; (b) conducting the biological assay with a compoundof the invention to provide a second assay value; wherein step (a) isconducted either before, after or concurrently with step (b); and (c)comparing the first assay value from step (a) with the second assayvalue from step (b). Exemplary biological assays include an AT₁ receptorbinding assay and a NEP enzyme inhibition assay.

Pharmaceutical Compositions and Formulations

Compounds of the invention are typically administered to a patient inthe form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to, oral,rectal, vaginal, nasal, inhaled, topical (including transdermal),ocular, and parenteral modes of administration. Further, the compoundsof the invention may be administered, for example orally, in multipledoses per day (for example, two, three, or four times daily), in asingle daily dose or a single weekly dose. It will be understood thatany form of the compounds of the invention, (that is, free base, freeacid, pharmaceutically acceptable salt, solvate, etc.) that is suitablefor the particular mode of administration can be used in thepharmaceutical compositions discussed herein.

Accordingly, in one embodiment, the invention relates to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of the invention. The compositions may containother therapeutic and/or formulating agents if desired. When discussingcompositions, the “compound of the invention” may also be referred toherein as the “active agent,” to distinguish it from other components ofthe formulation, such as the carrier. Thus, it is understood that theterm “active agent” includes compounds of formula I as well aspharmaceutically acceptable salts, solvates and prodrugs of thatcompound.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the invention. Thoseskilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,such as in bulk compositions, or less than a therapeutically effectiveamount, that is, individual unit doses designed for multipleadministration to achieve a therapeutically effective amount. Typically,the composition will contain from about 0.01-95 wt % of active agent,including, from about 0.01-30 wt %, such as from about 0.01-10 wt %,with the actual amount depending upon the formulation itself, the routeof administration, the frequency of dosing, and so forth. In oneembodiment, a composition suitable for an oral dosage form, for example,may contain about 5-70 wt %, or from about 10-60 wt % of active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with a pharmaceuticallyacceptable carrier and one or more optional ingredients. The resultinguniformly blended mixture may then be shaped or loaded into tablets,capsules, pills, canisters, cartridges, dispensers and the like usingconventional procedures and equipment.

In formulations where the compound of the invention contains a thiolgroup, additional consideration may be given to minimize or eliminateoxidation of the thiol to form a disulfide. In solid formulations, thismay be accomplished by reducing the drying time, decreasing the moisturecontent of the formulation, and including materials such as ascorbicacid, sodium ascorbate, sodium sulfite and sodium bisulfate, as well asmaterials such as a mixture of lactose and microcrystalline cellulose.In liquid formulations, stability of the thiol may be improved by theaddition of amino acids, antioxidants, or a combination of disodiumedetate and ascorbic acid.

In one embodiment, the pharmaceutical compositions are suitable for oraladministration. Suitable compositions for oral administration may be inthe form of capsules, tablets, pills, lozenges, cachets, dragees,powders, granules; solutions or suspensions in an aqueous or non-aqueousliquid; oil-in-water or water-in-oil liquid emulsions; elixirs orsyrups; and the like; each containing a predetermined amount of theactive agent.

When intended for oral administration in a solid dosage form (capsules,tablets, pills and the like), the composition will typically comprisethe active agent and one or more pharmaceutically acceptable carriers,such as sodium citrate or dicalcium phosphate. Solid dosage forms mayalso comprise: fillers or extenders, such as starches, microcrystallinecellulose, lactose, sucrose, glucose, mannitol, and/or silicic acid;binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; humectants, such as glycerol;disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as cetyl alcohol and/or glycerol monostearate; absorbents, such askaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodiumsulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, optionally with one ormore of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (for example, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.Suspensions may contain suspending agents such as, for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminium metahydroxide, bentonite,agar-agar and tragacanth, and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing apatient, that is, each unit containing a predetermined quantity of theactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

In another embodiment, the compositions of the invention are suitablefor inhaled administration, and will typically be in the form of anaerosol or a powder. Such compositions are generally administered usingwell-known delivery devices, such as a nebulizer, dry powder, ormetered-dose inhaler. Nebulizer devices produce a stream of highvelocity air that causes the composition to spray as a mist that iscarried into a patient's respiratory tract. An exemplary nebulizerformulation comprises the active agent dissolved in a carrier to form asolution, or micronized and combined with a carrier to form a suspensionof micronized particles of respirable size. Dry powder inhalersadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. An exemplary dry powderformulation comprises the active agent dry-blended with an excipientsuch as lactose, starch, mannitol, dextrose, polylactic acid,polylactide-co-glycolide, and combinations thereof. Metered-doseinhalers discharge a measured amount of the active agent usingcompressed propellant gas. An exemplary metered-dose formulationcomprises a solution or suspension of the active agent in a liquefiedpropellant, such as a chlorofluorocarbon or hydrofluoroalkane. Optionalcomponents of such formulations include co-solvents, such as ethanol orpentane, and surfactants, such as sorbitan trioleate, oleic acid,lecithin, glycerin, and sodium lauryl sulfate. Such compositions aretypically prepared by adding chilled or pressurized hydrofluoroalkane toa suitable container containing the active agent, ethanol (if present)and the surfactant (if present). To prepare a suspension, the activeagent is micronized and then combined with the propellant.Alternatively, a suspension formulation can be prepared by spray dryinga coating of surfactant on micronized particles of the active agent. Theformulation is then loaded into an aerosol canister, which forms aportion of the inhaler.

Compounds of the invention can also be administered parenterally (forexample, by subcutaneous, intravenous, intramuscular, or intraperitonealinjection). For such administration, the active agent is provided in asterile solution, suspension, or emulsion. Exemplary solvents forpreparing such formulations include water, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, oils, gelatin,fatty acid esters such as ethyl oleate, and the like. Parenteralformulations may also contain one or more anti-oxidants, solubilizers,stabilizers, preservatives, wetting agents, emulsifiers, and dispersingagents. Surfactants, additional stabilizing agents or pH-adjustingagents (acids, bases or buffers) and anti-oxidants are particularlyuseful to provide stability to the formulation, for example, to minimizeor avoid hydrolysis of ester and amide linkages, or dimerization ofthiols that may be present in the compound. These formulations may berendered sterile by use of a sterile injectable medium, a sterilizingagent, filtration, irradiation, or heat. In one particular embodiment,the parenteral formulation comprises an aqueous cyclodextrin solution asthe pharmaceutically acceptable carrier. Suitable cyclodextrins includecyclic molecules containing six or more α-D-glucopyranose units linkedat the 1,4 positions by a linkages as in amylase, β-cyclodextrin orcycloheptaamylose. Exemplary cyclodextrins include cyclodextrinderivatives such as hydroxypropyl and sulfobutyl ether cyclodextrinssuch as hydroxypropyl-β-cyclodextrin and sulfobutyl etherβ-cyclodextrin. Exemplary buffers for such formulations includecarboxylic acid-based buffers such as citrate, lactate and maleatebuffer solutions.

Compounds of the invention can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, thecompound can be admixed with permeation enhancers, such as propyleneglycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and thelike, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

If desired, the compounds of the invention may be administered incombination with one or more other therapeutic agents. Thus, in oneembodiment, pharmaceutical compositions of the invention contain otherdrugs that are co-administered with a compound of the invention. Forexample, the composition may further comprise one or more drugs (alsoreferred to as “secondary agents(s)”) selected from the group ofdiuretics, β₁ adrenergic receptor blockers, calcium channel blockers,angiotensin-converting enzyme inhibitors, AT₁ receptor antagonists,neprilysin inhibitors, non-steroidal anti-inflammatory agents,prostaglandins, anti-lipid agents, anti-diabetic agents, anti-thromboticagents, renin inhibitors, endothelin receptor antagonists, endothelinconverting enzyme inhibitors, aldosterone antagonists,angiotensin-converting enzyme/neprilysin inhibitors, and combinationsthereof. Such therapeutic agents are well known in the art, and specificexamples are described herein. By combining a compound of the inventionwith a secondary agent, triple therapy can be achieved; AT₁ receptorantagonist activity, NEP inhibition activity, and activity associatedwith the secondary agent (for example, β₁ adrenergic receptor blocker)can be achieved using only two active components. Since compositionscontaining two active components are typically easier to formulate thancompositions containing three active components, such two-componentcompositions provide a significant advantage over compositionscontaining three active components. Accordingly, in yet another aspectof the invention, a pharmaceutical composition comprises a compound ofthe invention, a second active agent, and a pharmaceutically acceptablecarrier. Third, fourth etc. active agents may also be included in thecomposition. In combination therapy, the amount of compound of theinvention that is administered, as well as the amount of secondaryagents, may be less than the amount typically administered inmonotherapy.

Compounds of the invention may be physically mixed with the secondactive agent to form a composition containing both agents; or each agentmay be present in separate and distinct compositions which areadministered to the patient simultaneously or at separate times. Forexample, a compound of the invention can be combined with a secondactive agent using conventional procedures and equipment to form acombination of active agents comprising a compound of the invention anda second active agent. Additionally, the active agents may be combinedwith a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition comprising a compound of the invention, a second activeagent and a pharmaceutically acceptable carrier. In this embodiment, thecomponents of the composition are typically mixed or blended to create aphysical mixture. The physical mixture is then administered in atherapeutically effective amount using any of the routes describedherein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the patient. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration of thecompound of the invention, ranging anywhere from concurrent withadministration of the compound of the invention to about 24 hourspost-dose. This is also referred to as sequential administration. Thus,a compound of the invention can be orally administered simultaneously orsequentially with another active agent using two tablets, with onetablet for each active agent, where sequential may mean beingadministered immediately after administration of the compound of theinvention or at some predetermined time later (for example, one hourlater or three hours later). Alternatively, the combination may beadministered by different routes of administration, that is, one orallyand the other by inhalation.

In one embodiment, the kit comprises a first dosage form comprising acompound of the invention and at least one additional dosage formcomprising one or more of the secondary agents set forth herein, inquantities sufficient to carry out the methods of the invention. Thefirst dosage form and the second (or third, etc,) dosage form togethercomprise a therapeutically effective amount of active agents for thetreatment or prevention of a disease or medical condition in a patient.

Secondary agent(s), when included, are present in a therapeuticallyeffective amount such that they are typically administered in an amountthat produces a therapeutically beneficial effect when co-administeredwith a compound of the invention. The secondary agent can be in the formof a pharmaceutically acceptable salt, solvate, optically purestereoisomer, and so forth. The secondary agent may also be in the formof a prodrug, for example, a compound having a carboxylic acid groupthat has been esterified. Thus, secondary agents listed herein areintended to include all such forms, and are commercially available orcan be prepared using conventional procedures and reagents.

In one embodiment, a compound of the invention is administered incombination with a diuretic. Representative diuretics include, but arenot limited to: carbonic anhydrase inhibitors such as acetazolamide anddichlorphenamide; loop diuretics, which include sulfonamide derivativessuch as acetazolamide, ambuside, azosemide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone, disulfamide,ethoxolamide, furosemide, mefruside, methazolamide, piretanide,torsemide, tripamide, and xipamide, as well as non-sulfonamide diureticssuch as ethacrynic acid and other phenoxyacetic acid compounds such astienilic acid, indacrinone and quincarbate; osmotic diuretics such asmannitol; potassium-sparing diuretics, which include aldosteroneantagonists such as spironolactone, and Na⁺ channel inhibitors such asamiloride and triamterene; thiazide and thiazide-like diuretics such asalthiazide, bendroflumethiazide, benzylhydrochlorothiazide,benzthiazide, buthiazide, chlorthalidone, chlorothiazide,cyclopenthiazide, cyclothiazide, epithiazide, ethiazide, fenquizone,flumethiazide, hydrochlorothiazide, hydroflumethiazide, indapamide,methylclothiazide, meticrane, metolazone, paraflutizide, polythiazide,quinethazone, teclothiazide, and trichloromethiazide; and combinationsthereof. In a particular embodiment, the diuretic is selected fromamiloride, bumetanide, chlorothiazide, chlorthalidone, dichlorphenamide,ethacrynic acid, furosemide, hydrochlorothiazide, hydroflumethiazide,indapamide, methylclothiazide, metolazone, torsemide, triamterene, andcombinations thereof. The diuretic will be administered in an amountsufficient to provide from about 5-50 mg per day, more typically 6-25 mgper day, with common dosages being 6.25 mg, 12.5 mg or 25 mg per day.

Compounds of the invention may also be administered in combination witha β₁ adrenergic receptor blocker. Representative β₁ adrenergic receptorblockers include, but are not limited to, acebutolol, alprenolol,amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol,bisoprolol, bopindolol, bucindolol, bucumolol, bufetolol, bufuralol,bunitrolol, bupranolol, bubridine, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol,esmolol, indenolol, labetolol, levobunolol, mepindolol, metipranolol,metoprolol such as metoprolol succinate and metoprolol tartrate,moprolol, nadolol, nadoxolol, nebivalol, nipradilol, oxprenolol,penbutolol, perbutolol, pindolol, practolol, pronethalol, propranolol,sotalol, sufinalol, talindol, tertatolol, tilisolol, timolol,toliprolol, xibenolol, and combinations thereof. In one particularembodiment, the β₁ adrenergic receptor blocker is selected fromatenolol, bisoprolol, metoprolol, propranolol, sotalol, and combinationsthereof.

In one embodiment, a compound of the invention is administered incombination with a calcium channel blocker. Representative calciumchannel blockers include, but are not limited to, amlodipine, anipamil,aranipine, barnidipine, bencyclane, benidipine, bepridil, clentiazem,cilnidipine, cinnarizine, diltiazem, efonidipine, elgodipine, etafenone,felodipine, fendiline, flunarizine, gallopamil, isradipine, lacidipine,lercanidipine, lidoflazine, lomerizine, manidipine, mibefradil,nicardipine, nifedipine, niguldipine, niludipine, nilvadipine,nimodipine, nisoldipine, nitrendipine, nivaldipine, perhexyline,prenylamine, ryosidine, scmotiadil, terodiline, tiapamil, verapamil, andcombinations thereof. In a particular embodiment, the calcium channelblocker is selected from amlodipine, bepridil, diltiazem, felodipine,isradipine, lacidipine, nicardipine, nifedipine, niguldipine,niludipine, nimodipine, nisoldipine, ryosidine, verapamil, andcombinations thereof.

Compounds of the invention can also be administered in combination withan angiotensin-converting enzyme (ACE) inhibitor. Representative ACEinhibitors include, but are not limited to, accupril, alacepril,benazepril, benazeprilat, captopril, ceranapril, cilazapril, delapril,enalapril, enalaprilat, fosinopril, fosinoprilat, imidapril, lisinopril,moexipril, monopril, moveltopril, pentopril, perindopril, quinapril,quinaprilat, ramipril, ramipril at, saralasin acetate, spirapril,temocapril, trandolapril, zofenopril, and combinations thereof. In aparticular embodiment, the ACE inhibitor is selected from: benazepril,enalapril, lisinopril, ramipril, and combinations thereof.

In one embodiment, a compound of the invention is administered incombination with an AT₁ receptor antagonist, also known as angiotensinII type 1 receptor blockers (ARBs). Representative ARBs include, but arenot limited to, abitesartan, benzyllosartan, candesartan, candesartancilexetil, elisartan, embusartan, enoltasosartan, eprosartan, fonsartan,forasartan, glycyllosartan, irbesartan, isoteoline, losartan, medoximil,milfasartan, olmesartan, opomisartan, pratosartan, ripisartan,saprisartan, saralasin, sarmesin, tasosartan, telmisartan, valsartan,zolasartan, and combinations thereof. In a particular embodiment, theARB is selected from candesartan, eprosartan, irbesartan, losartan,olmesartan, irbesartan, saprisartan, tasosartan, telmisartan, andcombinations thereof. Exemplary salts include eprosartan mesylate,losartan potassium salt, and olmesartan medoxomil. Typically, the ARBwill be administered in an amount sufficient to provide from about 4-600mg per dose, with exemplary daily dosages ranging from 20-320 mg perday.

In another embodiment, a compound of the invention is administered incombination with a neprilysin (NEP) inhibitor. Representative NEPinhibitors include, but are not limited to: candoxatril; candoxatrilat;dexecadotril ((+)-N-[2(R)-(acetylthiomethyl)-3-phenylpropionyl]glycinebenzyl ester); CGS-24128(3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-24592((S)-3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-25155 (N-[9(R)-(acetylthiomethyl)-10-oxo-1-azacyclodecan-2(S)-ylcarbonyl]-4 (R)-hydroxy-L-proline benzyl ester);3-(1-carbamoylcyclohexyl)propionic acid derivatives described in WO2006/027680 to Hepworth et al. (Pfizer Inc.); JMV-390-1(2(R)-benzyl-3-(N-hydroxycarbamoyl)propionyl-L-isoleucyl-L-leucine);ecadotril; phosphoramidon; retrothiorphan; RU-42827(2-(mercaptomethyl)-N-(4-pyridinyl)benzenepropionamide); RU-44004(N-(4-morpholinyl)-3-phenyl-2-(sulfanylmethyl)propionamide); SCH-32615((S)—N—[N-(1-carboxy-2-phenylethyl)-L-phenylalanyl]-β-alanine) and itsprodrug SCH-34826((S)—N—[N-[1-[[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-β-alanine);sialorphin; SCH-42495(N-[2(S)-(acetylsulfanylmethyl)-3-(2-methylphenyl)propionyl]-L-methionineethyl ester); spinorphin; SQ-28132(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]leucine); SQ-28603(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-(3-alanine); SQ-29072(7-[[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]heptanoic acid);thiorphan and its prodrug racecadotril; UK-69578(cis-4-[[[1-[2-carboxy-3-(2-methoxyethoxy)propyl]cyclopentyl]carbonyl]amino]cyclohexanecarboxylicacid); UK-447,841(2-{1-[3-(4-chlorophenyl)propylcarbamoyl]-cyclopentylmethyl}-4-methoxybutyricacid); UK-505,749((R)-2-methyl-3-{1-[3-(2-methylbenzothiazol-6-yl)propylcarbamoyl]cyclopentyl}propionicacid); 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid and 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid ethyl ester (WO 2007/056546); daglutril[(3S,2′R)-3-{1-[2′-(ethoxycarbonyl)-4′-phenylbutyl]-cyclopentan-1-carbonylamino}-2,3,4,5-tetrahydro-2-oxo-1H-1-benzazepine-1-aceticacid] described in WO 2007/106708 to Khder et al. (Novartis AG); andcombinations thereof. In a particular embodiment, the NEP inhibitor isselected from candoxatril, candoxatrilat, CGS-24128, phosphoramidon,SCH-32615, SCH-34826, SQ-28603, thiorphan, and combinations thereof. TheNEP inhibitor will be administered in an amount sufficient to providefrom about 20-800 mg per day, with typical daily dosages ranging from50-700 mg per day, more commonly 100-600 or 100-300 mg per day.

In yet another embodiment, a compound of the invention is administeredin combination with a non-steroidal anti-inflammatory agent (NSAID).Representative NSAIDs include, but are not limited to: acemetacin,acetyl salicylic acid, alclofenac, alminoprofen, amfenac, amiprilose,amoxiprin, anirolac, apazone, azapropazone, benorilate, benoxaprofen,bezpiperylon, broperamole, bucloxic acid, carprofen, clidanac,diclofenac, diflunisal, diftalone, enolicam, etodolac, etoricoxib,fenbufen, fenclofenac, fenclozic acid, fenoprofen, fentiazac, feprazone,flufenamic acid, flufenisal, fluprofen, flurbiprofen, furofenac,ibufenac, ibuprofen, indomethacin, indoprofen, isoxcpac, isoxicam,ketoprofen, ketorolac, lofemizole, lornoxicam, meclofenamate,meclofenamic acid, mefenamic acid, meloxicam, mesalamine, miroprofen,mofebutazone, nabumetone, naproxen, niflumic acid, oxaprozin, oxpinac,oxyphenbutazone, phenylbutazone, piroxicam, pirprofen, pranoprofen,salsalate, sudoxicam, sulfasalazine, sulindac, suprofen, tenoxicam,tiopinac, tiaprofenic acid, tioxaprofen, tolfenamic acid, tolmetin,triflumidate, zidometacin, zomepirac, and combinations thereof. In aparticular embodiment, the NSAID is selected from etodolac,flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meloxicam,naproxen, oxaprozin, piroxicam, and combinations thereof.

In yet another embodiment, a compound of the invention is administeredin combination with an anti-lipid agent. Representative anti-lipidagents include, but are not limited to, statins such as atorvastatin,fluvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin;cholesteryl ester transfer proteins (CETPs); and combinations thereof.

In yet another embodiment, a compound of the invention is administeredin combination with an anti-diabetic agent. Representative anti-diabeticagents include injectable drugs as well as orally effective drugs, andcombinations thereof. Examples of injectable drugs include, but are notlimited to, insulin and insulin derivatives. Examples of orallyeffective drugs include, but are not limited to: biguanides such asmetformin; glucagon antagonists; α-glucosidase inhibitors such asacarbose and miglitol; meglitinides such as repaglinide;oxadiazolidinediones; sulfonylureas such as chlorpropamide, glimepiride,glipizide, glyburide, and tolazamide; thiazolidinediones such aspioglitazone and rosiglitazone; and combinations thereof.

In one embodiment, a compound of the invention is administered incombination with an anti-thrombotic agent. Representativeanti-thrombotic agents include, but are not limited to, aspirin,anti-platelet agents, heparin, and combinations thereof. Compounds ofthe invention may also be administered in combination with a renininhibitor, examples of which include, but are not limited to, aliskiren,enalkiren, remikiren, and combinations thereof. In another embodiment, acompound of the invention is administered in combination with anendothelin receptor antagonist, representative examples of whichinclude, but are not limited to, bosentan, darusentan, tezosentan, andcombinations thereof. Compounds of the invention may also beadministered in combination with an endothelin converting enzymeinhibitor, examples of which include, but are not limited to,phosphoramidon, CGS 26303, and combinations thereof. In yet anotherembodiment, a compound of the invention is administered in combinationwith an aldosterone antagonist, representative examples of whichinclude, but are not limited to, eplerenone, spironolactone, andcombinations thereof.

Combined therapeutic agents may also be helpful in further combinationtherapy with compounds of the invention. For example, a combination ofthe ACE inhibitor enalapril (in the maleate salt form) and the diuretichydrochlorothiazide, which is sold under the mark Vaseretic®, or acombination of the calcium channel blocker amlodipine (in the besylatesalt form) and the ARB olmesartan (in the medoxomil prodrug form), or acombination of a calcium channel blocker and a statin, all may also beused with the compounds of the invention. Dual-acting agents may also behelpful in combination therapy with compounds of the invention. Forexample, angiotensin-converting enzyme/neprilysin (ACE/NEP) inhibitorssuch as: AVE-0848((4S,7S,12bR)-7-[3-methyl-2(S)-sulfanylbutyramido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); AVE-7688 (ilepatril) and its parent compound; BMS-182657(2-[2-oxo-3(S)-[3-phenyl-2(S)-sulfanylpropionamido]-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]aceticacid); CGS-26303([N-[2-(biphenyl-4-yl)-1(S)-(1H-tetrazol-5-yl)ethyl]amino]methylphosphonicacid); CGS-35601(N-[1-[4-methyl-2(S)-sulfanylpentanamido]cyclopentylcarbonyl]-L-tryptophan);fasidotril; fasidotrilate; enalaprilat; ER-32935((3R,6S,9aR)-6-[3(S)-methyl-2(S)-sulfanylpentanamido]-5-oxoperhydrothiazolo[3,2-a]azepine-3-carboxylicacid); gempatrilat; MDL-101264((4S,7S,12bR)-7-[2(S)-(2-morpholinoacetylthio)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); MDL-101287([4S-[4α,7α(R*),12bβ]]-7-[2-(carboxymethyl)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); omapatrilat; RB-105(N-[2(S)-(mercaptomethyl)-3(R)-phenylbutyl]-L-alanine); sampatrilat;SA-898((2R,4R)—N-[2-(2-hydroxyphenyl)-3-(3-mercaptopropionyl)thiazolidin-4-ylcarbonyl]-L-phenylalanine);Sch-50690(N-[1(S)-carboxy-2-[N2-(methanesulfonyl)-L-lysylamino]ethyl]-L-valyl-L-tyrosine);and combinations thereof, may also be included. In one particularembodiment, the ACE/NEP inhibitor is selected from: AVE-7688,enalaprilat, fasidotril, fasidotrilate, omapatrilat, sampatrilat, andcombinations thereof.

Other therapeutic agents such as α₂-adrenergic receptor agonists andvasopressin receptor antagonists may also be helpful in combinationtherapy. Exemplary α₂-adrenergic receptor agonists include clonidine,dexmedetomidine, and guanfacine. Exemplary vasopressin receptorantagonists include tolvaptan.

The following formulations illustrate representative pharmaceuticalcompositions of the invention.

Exemplary Hard Gelatin Capsules for Oral Administration

A compound of the invention (50 g), 440 g spray-dried lactose and 10 gmagnesium stearate are thoroughly blended. The resulting composition isthen loaded into hard gelatin capsules (500 mg of composition percapsule). Alternately, a compound of the invention (20 mg) is thoroughlyblended with starch (89 mg), microcrystalline cellulose (89 mg) andmagnesium stearate (2 mg). The mixture is then passed through a No. 45mesh U.S. sieve and loaded into a hard gelatin capsule (200 mg ofcomposition per capsule).

Exemplary Gelatin Capsule Formulation for Oral Administration

A compound of the invention (100 mg) is thoroughly blended withpolyoxyethylene sorbitan monooleate (50 mg) and starch powder (250 mg).The mixture is then loaded into a gelatin capsule (300 mg of compositionper capsule).

Alternately, a compound of the invention (40 mg) is thoroughly blendedwith microcrystalline cellulose (Avicel PH 103; 260 mg) and magnesiumstearate (0.8 mg). The mixture is then loaded into a gelatin capsule(Size #1, White, Opaque) (300 mg of composition per capsule).

Exemplary Tablet Formulation for Oral Administration

A compound of the invention (10 mg), starch (45 mg) and microcrystallinecellulose (35 mg) are passed through a No. 20 mesh U.S. sieve and mixedthoroughly. The granules so produced are dried at 50-60° C. and passedthrough a No. 16 mesh U.S. sieve. A solution of polyvinylpyrrolidone (4mg as a 10% solution in sterile water) is mixed with sodiumcarboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg), and talc (1mg), and this mixture is then passed through a No. 16 mesh U.S. sieve.The sodium carboxymethyl starch, magnesium stearate and talc are thenadded to the granules. After mixing, the mixture is compressed on atablet machine to afford a tablet weighing 100 mg.

Alternately, a compound of the invention (250 mg) is thoroughly blendedwith microcrystalline cellulose (400 mg), silicon dioxide fumed (10 mg),and stearic acid (5 mg). The mixture is then compressed to form tablets(665 mg of composition per tablet).

Alternately, a compound of the invention (400 mg) is thoroughly blendedwith cornstarch (50 mg), croscarmellose sodium (25 mg), lactose (120mg), and magnesium stearate (5 mg). The mixture is then compressed toform a single-scored tablet (600 mg of composition per tablet).

Alternately, a compound of the invention (100 mg) is thoroughly blendedwith cornstarch (100 mg) with an aqueous solution of gelatin (20 mg).The mixture is dried and ground to a fine powder. Microcrystallinecellulose (50 mg) and magnesium stearate (5 mg) are then admixed withthe gelatin formulation, granulated and the resulting mixture compressedto form tablets (100 mg of the compound of the invention per tablet).

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of the compound of the invention per 10 mL of suspension:

Ingredients Amount Compound of the invention 1.0 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum ® K 1.0 g(magnesium aluminum silicate) Flavoring 0.035 mL Colorings 0.5 mgDistilled water q.s. to 100 mL

Exemplary Liquid Formulation for Oral Administration

A suitable liquid formulation is one with a carboxylic acid-based buffersuch as citrate, lactate and maleate buffer solutions. For example, acompound of the invention (which may be pre-mixed with DMSO) is blendedwith a 100 mM ammonium citrate buffer and the pH adjusted to pH 5, or isblended with a 100 mM citric acid solution and the pH adjusted to pH 2.Such solutions may also include a solubilizing excipient such as acyclodextrin, for example the solution may include 10 wt %hydroxypropyl-β-cyclodextrin.

Other suitable formulations include a 5% NaHCO₃ solution, with orwithout cyclodextrin.

Exemplary Injectable Formulation for Administration by Injection

A compound of the invention (0.2 g) is blended with 0.4 M sodium acetatebuffer solution (2.0 mL). The pH of the resulting solution is adjustedto pH 4 using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodiumhydroxide, as necessary, and then sufficient water for injection isadded to provide a total volume of 20 mL. The mixture is then filteredthrough a sterile filter (0.22 micron) to provide a sterile solutionsuitable for administration by injection.

Exemplary Compositions for Administration by Inhalation

A compound of the invention (0.2 mg) is micronized and then blended withlactose (25 mg). This blended mixture is then loaded into a gelatininhalation cartridge. The contents of the cartridge are administeredusing a dry powder inhaler, for example.

Alternately, a micronized compound of the invention (10 g) is dispersedin a solution prepared by dissolving lecithin (0.2 g) in demineralizedwater (200 mL). The resulting suspension is spray dried and thenmicronized to form a micronized composition comprising particles havinga mean diameter less than about 1.5 μm. The micronized composition isthen loaded into metered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 μgto about 500 μg of the compound of the invention per dose whenadministered by the inhaler.

Alternately, a compound of the invention (25 mg) is dissolved in citratebuffered (pH 5) isotonic saline (125 mL). The mixture is stirred andsonicated until the compound is dissolved. The pH of the solution ischecked and adjusted, if necessary, to pH 5 by slowly adding aqueous 1 NNaOH. The solution is administered using a nebulizer device thatprovides about 10 μg to about 500 μg of the compound of the inventionper dose.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard, generally accepted meaning.

-   -   ACE angiotensin converting enzyme    -   AcOH acetic acid    -   APP aminopeptidase P    -   AT₁ angiotensin II type 1 (receptor)    -   AT₂ angiotensin II type 2 (receptor)    -   BSA bovine serum albumin    -   DCM dichloromethane or methylene chloride    -   DIPEA N,N-diisopropylethylamine    -   DMF N,N-dimethylformamide    -   DMSO dimethyl sulfoxide    -   Dnp 2,4-dinitrophenyl    -   DOCA deoxycorticosterone acetate    -   EDC N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride    -   EDTA ethylenediaminetetraacetic acid    -   EtOAc ethyl acetate    -   HATU N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium        hexafluorophosphate

HOBt 1-hydroxybenzotriazole hydrate

-   -   LiHMDS lithium hexamethyl disilazide    -   Mca (7-methoxycoumarin-4-yl)acyl    -   MeCN acetonitrile    -   MeOH methanol    -   NBS N-bromosuccinimide    -   NEP neprilysin (EC 3.4.24.11)    -   PBS phosphate buffered saline    -   SHR spontaneously hypertensive rat    -   TFA trifluoroacetic acid    -   THF tetrahydrofuran    -   Tris tris(hydroxymethyl)aminomethane    -   Tween-20 polyethylene glycol sorbitan monolaurate

Unless noted otherwise, all materials, such as reagents, startingmaterials and solvents, were purchased from commercial suppliers (suchas Sigma-Aldrich, Fluka Riedel-de Haën, and the like) and were usedwithout further purification.

Reactions were run under nitrogen atmosphere, unless noted otherwise.The progress of reactions were monitored by thin layer chromatography(TLC), analytical high performance liquid chromatography (anal. HPLC),and mass spectrometry, the details of which are given in specificexamples. Solvents used in analytical HPLC were as follows: solvent Awas 98% H₂O/2% MeCN/1.0 mL/L TFA; solvent B was 90% MeCN/10% H₂O/1.0mL/L TFA.

Reactions were worked up as described specifically in each preparationor example; commonly reaction mixtures were purified by extraction andother purification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by preparative HPLC, typically using Microsorb C18and Microsorb BDS column packings and conventional eluents. Progress ofreactions was typically measured by liquid chromatography massspectrometry (LCMS). Characterization of isomers were done by NuclearOverhauser effect spectroscopy (NOE). Characterization of reactionproducts was routinely carried out by mass and ¹H-NMR spectrometry. ForNMR measurement, samples were dissolved in deuterated solvent (CD₃OD,CDCl₃, or DMSO-d₆), and ¹H-NMR spectra were acquired with a VarianGemini 2000 instrument (400 MHz) under standard observation conditions.Mass spectrometric identification of compounds was typically conductedusing an electrospray ionization method (ESMS) with an AppliedBiosystems (Foster City, Calif.) model API 150 EX instrument or anAgilent (Palo Alto, Calif.) model 1200 LC/MSD instrument.

Preparation 1 2-(1-Trityl-1H-tetrazol-5-yl)phenylboronic Acid

A solution of benzonitrile (60.0 g, 581.9 mmol, 1.0 equiv) in DMF(anhydrous) (600 mL) was placed in a flask that had been purged andmaintained with an inert atmosphere of nitrogen. Ammonium chloride (40.5g, 757.2 mmol, 1.3 equiv) was added, followed by the addition of lithiumchloride (2.0 g, 47.2 mmol, 0.1 equiv). To the mixture was addedazidosodium (49.4 g, 756.8 mmol, 1.3 equiv). The resulting solution wasstirred overnight, while maintaining the temperature at 100° C. Thesolids were filtered out and washed with EtOAc. The filtrates werecombined and concentrated in vacuo. The residue was dissolved in a 10%NaOH aqueous solution (350 ml). The resulting solution was extractedwith EtOAc (100 mL) and the aqueous layers were combined. The pH valueof the solution was adjusted to 2 with concentrated HCl. The solids werefiltered out and dried over reduced pressure to yield5-phenyl-1H-tetrazole (78.0 g) as a white solid.

ES m/z: [M+H]⁺ calcd for C₇H₆N₄, 147.1. found 147.1.

A solution of the tetrazole (50.0 g, 342.1 mmol, 1.0 equiv) in DCM(dried) (150 mL) was placed in a flask that had been purged andmaintained with an inert atmosphere of nitrogen. Triethylamine (45.0 g,444.7 mmol, 1.30 equiv) at 0° C. was added to the mixture, followed bythe addition of chlorotriphenylmethane (100.0 g, 358.7 mmol, 1.1 equiv)in several batches at 0° C. The resulting solution was stirred for 3hours at room temperature. The solids were collected by filtration, andthe filtrate cake was washed with cold EtOAc (1×100 mL) and water (3×300mL). The solids were dried under reduced pressure to yield5-phenyl-1-trityl-1H-tetrazole (125 g) as a white solid. ES m/z: [M+H]+calcd for C₂₆H₂₀N₄, 389.1. found 389.1.

A solution of the protected tetrazole (70.0 g, 180.2 mmol, 1.0 equiv) inTHF (dried) (560 mL) was placed in a flask that had been purged andmaintained with an inert atmosphere of nitrogen. The mixture was cooledto −25° C. Butyllithium was added to quench the trace of water in thereaction mixture until the color of the mixture remained red for atleast 5 minutes. Butyllithium (80.0 mL, 2.5 mol/L) was then addeddropwise to the mixture over a period of about 40 minutes at atemperature below −25° C. The temperature of the mixture was raised to−5° C., and the mixture was stirred at −5° C. for 3 hours. The mixturewas then cooled to −25° C. and triisopropyl borate (50.8 g, 270.1 mmol,1.5 equiv) was added dropwise. The mixture was warmed to temperature inthe range of 25-35° C. and stirred for 2 hours. The mixture was cooledagain in the range of 0-5° C., and 3% aqueous AcOH (470 mL) was slowlyadded over 30-40 minutes. The mixture was stirred for 30-40 minutes andthen filtered. The crude product was washed with water (2×300 mL) anddried over reduced pressure. The crude product was then purified byrecrystallization from EtOAc (1 g/15 ml) to yield the title compound (54g) as a white solid.

ES m/z: [M+H]⁺ calcd for C₂₆H₂₁BN₄O₂, 433.1. found 433.1.

¹H-NMR (400 MHz, DMSO, 400 ppm): δ(ppm)=7.96 (br, 2H), 7.84-7.86 (m,1H), 7.51-7.53 (m, 1H), 7.44-7.47 (m, 2H), 7.39-7.41 (m, 9H), 7.08-7.10(m, 6H).

Preparation 2 (Tetrazol-5-yl)phenylboronic Acid

2-(1-Trityl-1H-tetrazol-5-yl)phenylboronic acid (11.5 g, 26.6 mmol) wascombined with 1,4-dioxane (41.5 mL, 532.1 mmol) and 4 M of HCl in1,4-dioxane (13.3 mL, 53.2 mmol). The mixture was stirred for 2 hours.EtOAc (100 mL) was added. 10 M NaOH was added until pH˜9, with constantstirring. The organic layer was extracted and discarded. The aqueouslayer was acidified to pH˜2 with DCM (10 mL). The product crashed outand was filtered and dried to obtain the title compound (3.5 g) as awhite solid.

Preparation 3 4-(4-Bromobenzyl)-5-propyloxazole-2-carboxylic Acid MethylEster

A solution of t-butyl 2-aminoacetate (113.7 g, 866.8 mmol, 1.0 equiv) intoluene (1000 mL) was combined with benzophenone (157.9 g, 866.5 mmol,1.0 equiv) and 4-methylbenzenesulfonic acid (14.9 g, 86.5 mmol, 0.1equiv). The resulting solution was heated to reflux overnight. Themixture was then concentrated in vacuo to 600 mL. The mixture was cooledto 15° C. with a water/ice bath. The product precipitated and the solidswere collected by filtration. The solids was washed with petroleum ether(5×150 mL) to yield t-butyl 2-(diphenylmethyleneamino)acetate (125 g) asa white solid.

A solution of the acetate (80 g, 265.4 mmol, 1.0 equiv) in THF (500 mL)was placed in a flask that had been purged and maintained undernitrogen. This was followed by the addition of LiHMDS (268.3 mL, 1.1equiv, 1.1 M) dropwise with stirring at −30° C. The resulting solutionwas stirred for 4 hours at −30˜0° C. in a liquid nitrogen bath. To thiswas added a solution of 1-bromo-4-(bromomethyl)benzene (67.7 g, 270.9mmol, 1.0 equiv) in THF (200 mL) dropwise with stirring at −30° C. Theresulting solution was stirred for an additional 1 hour at roomtemperature. The reaction was then quenched by the addition of water(500 mL). The resulting solution was extracted with EtOAc and theorganic layers combined. The resulting mixture was washed with saturatedaqueous NaCl (2×300 mL). The mixture was dried over anhydrous Na₂SO₄ andconcentrated in vacuo to yield t-butyl3-(4-bromophenyl)-2-(diphenylmethyleneamino)propanoate (115 g) as ayellow solid. A solution of this solid (115 g, 148.6 mmol, 1.0 equiv) inHCl/H₂O (6N) (300 mL) was heated to reflux overnight. The resultingmixture was concentrated in vacuo and the crude product was washed withEtOAc to yield 2-amino-3-(4-bromophenyl)-propanoic acid hydrochloride(52.7 g) as a white solid.

A solution of the propanoic acid hydrochloride (52.7 g, 140.9 mmol, 1.0equiv) in THF (200 mL) was combined with a solution of NaOH (15 g, 375.0mmol, 2.0 equiv) in water (200 mL), and (Boc)₂O (43 g, 197.0 mmol, 1.1equiv). The resulting solution was stirred overnight at roomtemperature. The mixture was then concentrated in vacuo and the pHadjusted to 5 with AcOH. The resulting solution was extracted with EtOAcand the organic layers combined. The resulting mixture was washed withwater (1×300 mL) and saturated aqueous NaCl (1×300 mL), thenconcentrated in vacuo. The crude product was washed with petroleum etherto yield 3-(4-bromophenyl)-2-(tert-butoxycarbonyl)propanoic acid (45 g).

¹H-NMR (300 MHz, CDCl₃): δ(ppm)=8.37 (1H, s), 7.45 (2H, d, J=5 Hz), 7.08(2H, d, J=9 Hz), 4.97 (1H, d), 4.62 (1H, m), 3.02-3.22 (2H, d), 1.40(9H, s).

A solution of the propanoic acid (45 g, 117.7 mmol, 1.0 equiv) in DCM(400 mL) was combined with N-methoxymethanamine hydrochloride (15.3 g,156.9 mmol, 1.2 equiv), EDC.HCl (30.1 g, 157.0 mmol, 1.2 equiv), HOBT(21.2 g, 156.9 mmol, 1.2 equiv), and triethylamine (30 mL). Theresulting solution was stirred overnight at room temperature. Thereaction was then quenched by the addition of water (300 mL). Theresulting mixture was washed with 2M citric acid (2×200 mL) andsaturated NaHCO₃ (1×200 mL), then dried over anhydrous MgSO₄ andconcentrated in vacuo to yield t-butyl3-(4-bromophenyl)-1-(methoxy(methyl)amino)-1-oxopropan-2-ylcarbamate (26g) as a white solid.

A solution of propylmagnesium bromide (29.7 g, 201.6 mmol, 3.0 equiv) inTHF (200 mL) was added dropwise with stirring at 0° C. to a solution ofthe carbamate (26 g, 66.5 mmol, 1.0 equiv) in THF (200 mL). Theresulting solution was stirred overnight at room temperature. Thereaction was then quenched by the addition of aqueous NH₄Cl (200 mL).The resulting solution was extracted with EtOAc and the organic layerscombined. The resulting mixture was washed with saturated NaHCO₃ (200mL) and saturated aqueous NaCl (200 mL), then dried over anhydrousMgSO₄. The residue was applied onto a silica gel column with petroleumether/EtOAc (5:1) to yield t-butyl1-(4-bromophenyl)-3-oxohexan-2-ylcarbamate (14 g) as a white solid.

¹H-NMR (300 MHz, CDCl₃): δ(ppm)=7.43 (2H, d, J=6 Hz), 7.04 (2H, d, J=9Hz), 5.13 (1H, d), 4.53 (1H, m), 2.87-3.11 (2H, d), 2.42 (2H, t), 1.59(2H, m), 1.43 (9H, s), 0.9 (3H, t)

A solution of the carbamate (8.0 g, 21.2 mmol, 1.0 equiv) in ether (100mL) was placed in a flask, and HCL gas was introduced for 5 hours atroom temperature. The resulting mixture was concentrated in vacuo toyield crude 2-amino-1-(4-bromophenyl)hexan-3-one hydrochloride (7.2 g)as a white solid. A solution of this solid (7.2 g, 1.0 equiv) in THF(100 mL) and methyl 2-chloro-2-oxoacetate (2.9 g, 1.0 equiv) wascombined with triethylamine (4.7 g, 2.0 equiv), which was added dropwisewith stirring. The resulting solution was stirred overnight at roomtemperature. The reaction was then quenched by the addition of water(200 mL). The resulting solution was extracted with EtOAc and theorganic layers combined. The resulting mixture was washed with saturatedaqueous NaCl (1×100 mL) and dried over Na₂SO₄. The residue was appliedonto a silica gel column with petroleum ether/EtOAc (5:1) to yieldmethyl 2-(1-(4-bromophenyl)-3-oxohexan-2-ylamino)-2-oxoacetate (4.7 g)as a white solid.

A solution of the oxoacetate (4.7 g, 13.0 mmol, 1.0 equiv) in phosphoryltrichloride (30.3 g) was stirred for 2 hours at 130° C. in an oil bath.The reaction was then quenched by the addition of water/ice (100 mL).The pH was adjusted to 9 with sodium bicarbonate. The resulting solutionwas extracted with EtOAc and the organic layers combined. The resultingmixture was washed with saturated aqueous NaCl (2×200 mL), dried overanhydrous Na₂SO₄, and concentrated in vacuo. The residue was appliedonto a silica gel column with EtOAc/petroleum ether (1:10) to yield thetitle compound (3.2 g) as a light yellow solid. ES m/z: [M+H]⁺ 339.

¹H-NMR (300 MHz, CDCl₃): δ(ppm)=7.42 (2H, dd, J=3.3 Hz), 7.12 (2H, d,J=9 Hz), 3.96 (3H, s), 3.86 (2H, s), 2.64 (2H, t), 1.67 (2H, m), 0.91(3H, t)

Preparation 45-Propyl-4-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]oxazole-2-carboxylicAcid

A solution of 4-(4-bromobenzyl)-5-propyloxazole-2-carboxylic acid methylester (500.0 mg, 1474 mmol), (tetrazol-5-yl)phenylboronic acid (336 mg,1.8 mmol), tetrakis(triphenylphosphine)palladium(0) (85.2 mg, 73.7μmol), 1 M NaOH in water (4.7 mL, 4.7 mmol), and MeOH (7.2 mL, 177 mmol)was heated at 90° C. for 2 hours in the microwave. 6M aqueous HCl wasadded until pH 7 was achieved. The mixture was concentrated in vacuo.The concentrate was extracted with EtOAc (2×15 mL) and 1M aqueous HCl(10 mL). The combined organic extracts were dried over Na₂SO₄, filteredand concentrated in vacuo to yield the title compound (540 mg).

Compounds such as5-ethoxy-4-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]oxazole-2-carboxylicacid, as well as4-propyl-5-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]oxazole-2-carboxylicacid and4-ethoxy-5-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]oxazole-2-carboxylicacid can be prepared in a similar manner or by techniques that are wellknown in the art. For example, compounds where R³ is —O—C₁₋₅alkyl (e.g.,5-ethoxy-4-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]oxazole-2-carboxylicacid), can be made by the methods described in Thalhammer et al. (2009)Tetrahedron Letters 50:1045-1047 and Tussetschläger et al. (2006) Z.Naturforsch 61b:420-426.

Example 1(2R,3R)-2-Hydroxy-4-phenyl-3-({5-propyl-4-[2′-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-oxazole-2-carbonyl}amino)butyricAcid

A solution of5-propyl-4-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]oxazole-2-carboxylicacid (600 mg, 1 mmol1), (2R,3R)-3-amino-2-hydroxy-4-phenylbutyric acidethyl ester.HCl (560 mg, 2.2 mmol), HATU (820 mg, 2.2 mmol), DIPEA (939μL, 5.4 mmol) and DMF (8 mL) was prepared and stirred overnight. EtOAc(20 mL) and 1M aqueous HCl (10 mL) were added. The organic layer wasextracted, dried over Na₂SO₄, filtered and concentrated in vacuo toyield the crude intermediate, which was then dissolved in MeOH (5 mL)and 1M aqueous NaOH (5 mL). After 3 hours, the mixture was acidifiedwith AcOH (2 mL) and concentrated in vacuo. Reverse phase HPLC affordedthe title compound (30 mg; 92% purity) as well as(2S,3R)-2-hydroxy-4-phenyl-3-({5-propyl-4-[2′-(1h-tetrazol-5-yl)-biphenyl-4-ylmethyl]-oxazole-2-carbonyl}amino)butyricacid (4 mg; 96% purity). For both compounds, MS m/z: [M+H]⁺ calcd forC₃₁H₃₀N₆O₅, 567.23. found 567.6.

Example 2

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compoundsIIa-1 to IIa-18 and IIIa-1 to IIIa-18, having the following formula, canalso be prepared:

(IIa)

(IIIa)

# R R³ R⁴ R⁵  1 3-F ethoxy —CH₂SH benzyl  2 — propyl —CH₂SH i-butyl  32-F propyl —CH₂COOH 2-Cl-benzyl  4 — propyl —CH₂COOH 2-Br-benzyl  5 2-Fethoxy —CH₂COOH benzyl  6 2-F propyl —CH₂COOH 2-F-benzyl  7 — ethoxy—CH₂COOH 2-CH₃-benzyl  8 — propyl —CH₂COOH 2-CF₃-benzyl  9 3-F ethoxy—CH(OH)COOH benzyl 10 — propyl —CH(OH)C(O)OCH₃ benzyl 11 — propyl—CH(OH)COOH 2-Cl-benzyl 12 3,5-diF propyl —CH₂C(O)OCH₃ benzyl 13 3,5-diFethoxy —CH₂C(O)OCH₃ 2-Cl-benzyl 14 — ethoxy —CH₂C(O)OCH₃ 2-CH₃-benzyl 15— propyl —CH₂—N(OH)C(O)H benzyl 16 3,5-diF ethoxy —CH₂C(O)NH(OH) benzyl17 — propyl —CH₂C(O)NH(OH) 2-Cl-benzyl 18 — propyl —CH(OH)C(O)NH(OH)benzyl

Preparation 54-(2′-Acetylsulfamoylbiphenyl-4-ylmethyl)-5-propyloxazole-2-carboxylicacid

The title compound can be prepared in a manner similar to Preparation 4,using a protected sulfonamide, followed by a deprotection step.

Compounds such as4-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)-5-ethoxyoxazole-2-carboxylicacid,5-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)-4-propyloxazole-2-carboxylicacid, and5-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)-4-ethoxyoxazole-2-carboxylicacid can be prepared in a similar manner.

Example 3

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compoundsIIb-1 to IIb-18 and IIIb-1 to IIIb-18, having the following formula, canalso be prepared:

(IIb)

(IIIb)

# R R³ R⁴ R⁵  1 3-F ethoxy —CH₂SH benzyl  2 — propyl —CH₂SH i-butyl  32-F propyl —CH₂COOH 2-Cl-benzyl  4 — propyl —CH₂COOH 2-Br-benzyl  5 2-Fethoxy —CH₂COOH benzyl  6 2-F propyl —CH₂COOH 2-F-benzyl  7 — ethoxy—CH₂COOH 2-CH₃-benzyl  8 — propyl —CH₂COOH 2-CF₃-benzyl  9 3-F ethoxy—CH(OH)COOH benzyl 10 — propyl —CH(OH)C(O)OCH₃ benzyl 11 — propyl—CH(OH)COOH 2-Cl-benzyl 12 3,5-diF propyl —CH₂C(O)OCH₃ benzyl 13 3,5-diFethoxy —CH₂C(O)OCH₃ 2-Cl-benzyl 14 — ethoxy —CH₂C(O)OCH₃ 2-CH₃-benzyl 15— propyl —CH₂—N(OH)C(O)H benzyl 16 3,5-diF ethoxy —CH₂C(O)NH(OH) benzyl17 — propyl —CH₂C(O)NH(OH) 2-Cl-benzyl 18 — propyl —CH(OH)C(O)NH(OH)benzyl

Example 4

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compoundsIIc-1 to IIc-21 and IIIc-1 to IIIc-21, having the following formula, canalso be prepared:

(IIc)

(IIIc)

# R^(1a) R R³ R⁴ R⁵  1 —NH(CH₃) 2-F ethoxy —CH(OH)COOH benzyl  2—N(CH₃)₂ — propyl —CH(OH)COOH benzyl  3 —NH(CH₂CH₃) — ethoxy —CH(OH)COOHbenzyl  4 ethyl 3-F propyl —CH(OH)COOH benzyl  5 cyclopropyl — ethoxy—CH(OH)COOH benzyl  6 phenyl — propyl —CH(OH)COOH benzyl  7 2F-phenyl3,5-diF ethoxy —CH(OH)COOH benzyl  8 4-pyridyl 2-F propyl —CH(OH)COOHbenzyl  9 -3-isoxazolyl- — ethoxy —CH(OH)COOH benzyl 5-methyl 10-5-isoxazolyl — propyl —CH(OH)COOH benzyl 11 —OCH₂CH₃ 3-F ethoxy—CH(OH)COOH benzyl 12 —CH₂OCH₃ — propyl —CH(OH)COOH benzyl 13 —CH₂OCH₃ —ethoxy —CH₂COOH 2-Cl- benzyl 14 —(CH₂)₂—OCH₃ 3,5-diF propyl —CH(OH)COOHbenzyl 15 2-pyridyl 2-F ethoxy —CH(OH)COOH benzyl 16 —CH₂OH — propyl—CH(OH)COOH benzyl 17 —CH(CH₃)OH — ethoxy —CH(OH)COOH benzyl 18—C(CH₃)₂OH 3-F propyl —CH(OH)COOH benzyl 19 —C(CH₃)₂NH₂ — ethoxy—CH(OH)COOH benzyl 20 1-pyrrolidyl — propyl —CH(OH)COOH benzyl 214-morpholinyl 3,5-diF ethoxy —CH(OH)COOH benzyl

Preparation 65-Propyl-4-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}oxazole-2-carboxylicacid

The title compound can be prepared as follows: A solution of(tetrazol-5-yl)phenylboronic acid (1.0 mmol),4-(6-bromopyridin-3-ylmethyl)-5-propyloxazole-2-carboxylic acid ethylester (809 μmol), tetrakis(triphenylphosphine)palladium(0) (40 μmol),1.0 M NaOH in water (mmol) and MeOH (100 mmol) is sparged shortly,capped under nitrogen and heated in the microwave at 90° C. for 2 hours.The mixture is then filtered to remove the Pd (rinsed with MeOH). Thefiltrate is then concentrated to remove the McOH, then extracted withEtOAc. The organic layer is extracted with 1N NaOH. The combined aqueouslayers are acidified with 1 N HCl to pH 3-4 and extracted with EtOAc.The organics are dried over MgSO₄, filtered and concentrated to yieldthe title compound, which can be used without further purification.

Other compounds, such as4-propyl-5-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}oxazole-2-carboxylicacid,5-ethoxy-4-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}oxazole-2-carboxylicacid, and4-ethoxy-5-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}oxazole-2-carboxylicacid, can be prepared in a similar manner.

Example 5

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compoundsIId-1 to IId-22 and IIId-1 to IIId-22, having the following formula, canalso be prepared:

(IId)

(IIId)

# R¹ R³ R⁴ R⁵  1 1H-tetrazol-5-yl propyl —CH₂COOH 2-Cl-benzyl  21H-tetrazol-5-yl ethoxy —CH₂COOH 2-Cl-benzyl  3 1H-tetrazol-5-yl propyl—CH₂COOH 2-CF₃-benzyl  4 1H-tetrazol-5-yl ethoxy —CH₂COOH 2-CF₃-benzyl 5 1H-tetrazol-5-yl propyl —CH₂COOH 2-F-benzyl  6 1H-tetrazol-5-ylethoxy —CH₂COOH 2-F-benzyl  7 1H-tetrazol-5-yl propyl —CH(OH)COOH benzyl 8 1H-tetrazol-5-yl propyl —CH₂SH benzyl  9 1H-tetrazol-5-yl propyl—CH₂COOH 2-Br-benzyl 10 1H-tetrazol-5-yl propyl —CH(OH)COOH 2-Cl-benzyl11 1H-tetrazol-5-yl propyl —CH₂COOH benzyl 12 1H-tetrazol-5-yl propyl—CH₂COOH i-butyl 13 1H-tetrazol-5-yl propyl —CH(OH)COOH i-butyl 141H-tetrazol-5-yl propyl —CH(OH)COOH benzyl 15 1H-tetrazol-5-yl propyl—CH₂COOH

16 1H-tetrazol-5-yl propyl —CH₂COOH

17 1H-tetrazol-5-yl propyl —CH₂COOH 3-F-benzyl 18 1H-tetrazol-5-ylpropyl —CH₂COOH 4-F-benzyl 19 —SO₂NH—C(O)CH₃ propyl —CH(OH)COOH benzyl20 —SO₂NH—C(O)CH₃ propyl —CH₂SH benzyl 21 —SO₂NH—C(O)CH₃ propyl —CH₂COOH2-Cl-benzyl 22 —SO₂NH—C(O)CH₃ propyl —CH₂COOH benzyl

Example 6

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compoundsIIe-1 to IIe-4 and IIIe-1 to IIIe-4, having the following formula, canalso be prepared:

(IIe)

(IIIe)

# R¹ R³ R⁴ R⁵ 1 1H-tetrazol-5-yl propyl —CH(OH)COOH benzyl 21H-tetrazol-5-yl propyl —CH₂COOH 2-Cl-benzyl 3 —SO₂NH—C(O)CH₃ propyl—CH(OH)COOH benzyl 4 —SO₂NH—C(O)CH₃ propyl —CH₂COOH 2-Cl-benzyl

Example 7

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compoundsIIf-1 to IIf-7 and IIIf-1 to IIIf-7, having the following formula, canalso be prepared:

(IIf)

(IIIf)

# R¹ R³ R⁴ R⁵ 1

propyl —CH₂SH benzyl 2

propyl —CH(OH)COOH benzyl 3

propyl —CH(OH)COOH benzyl 4

propyl —CH₂COOH benzyl 5

propyl —CH₂COOH 2-Cl- benzyl 6

propyl —CH(OH)COOH 2-Cl- benzyl 7

ethoxy —CH(OH)COOH benzyl

Example 8

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compoundsIIg-1 to IIg-6 and IIIg-1 to IIIg-6, having the following formula, canalso be prepared:

(IIg)

(IIIg)

# R¹ R³ R⁴ R⁵ 1 1H-tetrazol-5-yl propyl —CH₂COOH benzyl 21H-tetrazol-5-yl propyl —CH₂COOH 2-Cl-benzyl 3 1H-tetrazol-5-yl propyl—CH(OH)COOH benzyl 4 —SO₂NHC(O)CH₃ propyl —CH₂COOH benzyl 5—SO₂NHC(O)CH₃ propyl —CH(OH)COOH benzyl 6 —SO₂NHC(O)CH₃ propyl —CH₂COOH2-Cl-benzyl

Example 9

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compoundsIIh-1 and IIIh-1, having the following formula, can also be prepared:

(IIh)

(IIIh)

# R¹ R³ R⁴ R⁵ 1 1H-tetrazol-5-yl propyl —CH₂COOH 2-Cl-benzyl 11H-tetrazol-5-yl propyl —CH(OH)COOH benzyl

Assay 1 AT_(L) and AT₂ Radioligand Binding Assays

These in vitro assays were used to assess the ability of test compoundsto bind to the AT₁ and the AT₂ receptors.

Membrane Preparation from Cells Expressing Human AT₁ or AT₂ Receptors

Chinese hamster ovary (CHO-K1) derived cell lines stably expressing thecloned human AT₁ or AT₂ receptors, respectively, were grown in HAM's-F12medium supplemented with 10% fetal bovine serum, 10 μg/mlpenicillin/streptomycin, and 500 μg/ml geneticin in a 5% CO₂ humidifiedincubator at 37° C. AT₂ receptor expressing cells were grown in theadditional presence of 100 nM PD123,319 (AT₂ antagonist). When culturesreached 80-95% confluence, the cells were washed thoroughly in PBS andlifted with 5 mM EDTA. Cells were pelleted by centrifugation and snapfrozen in MeOH-dry ice and stored at −80° C. until further use.

For membrane preparation, cell pellets were resuspended in lysis buffer(25 mM Tris/HCl pH 7.5 at 4° C., 1 mM EDTA, and one tablet of CompleteProtease Inhibitor Cocktail Tablets with 2 mM EDTA per 50 mL buffer(Roche cat. #1697498, Roche Molecular Biochemicals, Indianapolis, Ind.))and homogenized using a tight-fitting Dounce glass homogenizer (10strokes) on ice. The homogenate was centrifuged at 1000×g, thesupernatant was collected and centrifuged at 20,000×g. The final pelletwas resuspended in membrane buffer (75 mM Tris/HCl pH 7.5, 12.5 mMMgCl₂, 0.3 mM EDTA, 1 mM EGTA, 250 mM sucrose at 4° C.) and homogenizedby extrusion through a 20G gauge needle. Protein concentration of themembrane suspension was determined by the method described in Bradford(1976) Anal Biochem. 72:248-54. Membranes were snap frozen in MeOH-dryice and stored at −80° C. until further use.

Ligand Binding Assay to Determine Compound Affinities for the Human AT₁and AT₂ Angiotensin Receptors

Binding assays were performed in 96-well Acrowell filter plates (PallInc., cat. #5020) in a total assay volume of 100 μL with 0.2 μg membraneprotein for membranes containing the human AT₁ receptor, or 2 g membraneprotein for membranes containing the human AT₂ receptor in assay buffer(50 mM Tris/HCl pH 7.5 at 20° C., 5 mM MgCl₂, 25 μM EDTA, 0.025% BSA).Saturation binding studies for determination of K_(d) values of theligand were done using N-terminally Europium-labeled angiotensin-II([Eu]AngII, H—(Eu—N¹)-Ahx-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-OH;PerkinElmer, Boston, Mass.) at 8 different concentrations ranging from0.1 nM to 30 nM. Displacement assays for determination of pK_(i) valuesof test compounds were done with [Eu]AngII at 2 nM and 11 differentconcentrations of drug ranging from 1 pM to 10 μM. Drugs were dissolvedto a concentration of 1 mM in DMSO and from there serially diluted intoassay buffer. Nonspecific binding was determined in the presence of 10μM unlabeled angiotensin-II. Assays were incubated for 120 minutes inthe dark, at room temperature or 37° C., and binding reactions wereterminated by rapid filtration through the Acrowell filter platesfollowed by three washes with 200 μL ice cold wash buffer (50 mMTris/HCl pH 7.5 at 4° C., 5 mM MgCl₂) using a Waters filtrationmanifold. Plates were tapped dry and incubated with 50 μl DELFIAEnhancement Solution (PerkinElmer cat. #4001-0010) at room temperaturefor 5 minutes on a shaker. Filter-bound [Eu]AngII was quantitatedimmediately on a Fusion plate reader (PerkinElmer) using Time ResolvedFluorescence (TRF). Binding data were analyzed by nonlinear regressionanalysis with the GraphPad Prism Software package (GraphPad Software,Inc., San Diego, Calif.) using the 3-parameter model for one-sitecompetition. The BOTTOM (curve minimum) was fixed to the value fornonspecific binding, as determined in the presence of 10 μM angiotensinII. K, values for drugs were calculated from observed IC₅₀ values andthe K_(d) value of [Eu]AngII according to the Cheng-Prusoff equationdescribed in Cheng et al. (1973) Biochem Pharmacol. 22(23):3099-108.Selectivities of test compounds for the AT₁ receptor over the AT₂receptor were calculated as the ratio of AT₂K_(i)/AT₁K_(i). Bindingaffinities of test compounds were expressed as negative decadiclogarithms of the K_(i) values (pK_(i)).

In this assay, a higher pK_(i) value indicates that the test compoundhas a higher binding affinity for the receptor tested. Compounds of theinvention tested in this assay are expected to have a pK_(i) at the AT₁receptor greater than or equal to about 7.0, for example, the compoundof Example 1 has a pK_(i) at the AT₁ receptor greater than 7.5.

Assay 2 In Vitro Assays for the Quantitation of Inhibitor Potencies(IC₅₀) at Human and Rat NEP, and Human ACE

The inhibitory activities of compounds at human and rat NEP and humanACE were determined using in vitro assays as described below.

Extraction of NEP Activity from Rat Kidneys

Rat NEP was prepared from the kidneys of adult Sprague Dawley rats.Whole kidneys were washed in cold PBS and brought up in ice-cold lysisbuffer (1% Triton X-114, 150 mM NaCl, 50 mM Tris pH 7.5; Bordier (1981)J. Biol. Chem. 256:1604-1607) in a ratio of 5 mL of buffer for everygram of kidney. Samples were homogenized using a polytron hand heldtissue grinder on ice. Homogenates were centrifuged at 1000×g in aswinging bucket rotor for 5 minutes at 3° C. The pellet was resuspendedin 20 mL of ice cold lysis buffer and incubated on ice for 30 minutes.Samples (15-20 mL) were then layered onto 25 mL of ice-cold cushionbuffer (6% w/v sucrose, 50 mM pH 7.5 Tris, 150 mM NaCl, 0.06%, TritonX-114), heated to 37° C. for 3-5 minutes and centrifuged at 1000×g in aswinging bucket rotor at room temperature for 3 minutes. The two upperlayers were aspirated off, leaving a viscous oily precipitate containingthe enriched membrane fraction. Glycerol was added to a concentration of50% and samples were stored at −20° C. Protein concentrations werequantitated using a BCA detection system with BSA as a standard.

Enzyme Inhibition Assays

Recombinant human NEP and recombinant human ACE were obtainedcommercially (R&D Systems, Minneapolis, Minn., catalog numbers 1182-ZNand 929-ZN, respectively). The fluorogenic peptide substrate Mca-BK2(Mca-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(Dnp)-OH; Johnson et al. (2000)Anal. Biochem. 286: 112-118) was used for the human NEP and ACE assays,and Mca-RRL (Mca-DArg-Arg-Leu-(Dnp)-OH; Medeiros et al. (1997) Braz. J.Med. Biol. Res. 30:1157-1162) was used for the rat NEP assay (both fromAnaspec, San Jose, Calif.).

The assays were performed in 384-well white opaque plates at roomtemperature using the respective fluorogenic peptides at a concentrationof 10 μM in assay buffer (50 mM Tris/HCl at 25° C., 100 mM NaCl, 0.01%Tween-20, 1 μM Zn, 0.025% BSA). Human NEP and human ACE were used atconcentrations that resulted in quantitative proteolysis of 5 μM ofMca-BK2 within 20 minutes at room temperature. The rat NEP enzymepreparation was used at a concentration that yielded quantitativeproteolysis of 3 μM of Mca-RRL within 20 minutes at room temperature.

Test compounds were diluted to 12 concentrations from 10 μM to 20 μM inAssay Buffer. Assays were started by adding 25 μL of enzyme to 12.54 oftest compound at each of the 12 concentrations. Test compounds wereallowed to equilibrate with the enzyme for 10 minutes before 12.5 μL ofthe fluorogenic substrates were added to initiate the reaction.Reactions were terminated by the addition of 10 μL of 3.6% glacialacetic acid after 20 minutes of incubation.

For sulfhydryl-containing test compounds, the test compounds may bediluted in Assay Buffer containing a 400 μM concentration oftris(2-carboxyethyl)phosphine hydrochloride (Thermo Scientific,Rockford, Ill.) (TCEP). The test compounds are then allowed to reducefor 40 minutes at room temperature before adding the enzyme. Testcompounds are then allowed to equilibrate with the enzyme for 20 minutesbefore adding the fluorogenic substrates. Reactions are terminated asabove.

Plates were read on a fluorometer with excitation and emissionwavelengths set to 320 nm and 405 nm, respectively. Raw data (relativefluorescence units) were normalized to % activity from the average highreadings (no inhibition, 100% enzyme activity) and average low readings(full inhibition, highest inhibitor concentration, 0% enzyme activity)using three standard NEP and ACE inhibitors, respectively. Nonlinearregression of the normalized data was performed using a one sitecompetition model (GraphPad Software, Inc., San Diego, Calif.). Datawere reported as pIC₅₀ values. Compounds of the invention tested in thisassay are expected to have a pIC₅₀ for the NEP enzyme greater than orequal to about 6.0, the exception being those compounds that areprodrugs. For example, the compound of Example 1 has a pIC₅₀ for the NEPenzyme greater than 6.0.

Assay 3 Pharmacodynamic (PD) Assay for ACE, AT₁, and NEP Activity inAnesthetized Rats

Male, Sprague Dawley, normotensive rats are anesthetized with 120 mg/kg(i.p.) of inactin. Once anesthetized, the jugular vein, carotid artery(PE 50 tubing) and bladder (URI-1 urinary silicone catheter) arecannulated and a tracheotomy is performed (Teflon Needle, size 14 gauge)to faciliate spontaneous respiration. The animals are then allowed a 60minute stablization period and kept continuously infused with 5 mL/kg/hof saline (0.9%) throughout, to keep them hydrated and ensure urineproduction. Body temperature is maintained throughout the experiment byuse of a heating pad. At the end of the 60 minute stabilization period,the animals are dosed intravenously (i.v.) with two doses of angiotensin(AngI, 1.0 μg/kg, for ACE inhibitor activity; AngII, 0.1 μg/kg, for AT₁receptor antagonist activity) at 15 minutes apart. At 15 minutespost-second dose of angiotensin (AngI or AngII), the animals are treatedwith vehicle or test compound. Five minutes later, the animals areadditionally treated with a bolus i.v. injection of atrial natriureticpeptide (ANP; 30 μg/kg). Urine collection (into pre-weighted eppendorftubes) is started immediately after the ANP treatment and continued for60 minutes. At 30 and 60 minutes into urine collection, the animals arere-challenged with angiotensin (AngI or AngII). Blood pressuremeasurements are done using the Notocord system (Kalamazoo, Mich.).Urine samples are frozen at −20° C. until used for the cGMP assay. UrinecGMP concentrations are determined by Enzyme Immuno Assay using acommercial kit (Assay Designs, Ann Arbor, Mich., Cat. No. 901-013).Urine volume is determined gravimetrically. Urinary cGMP output iscalculated as the product of urine output and urine cGMP concentration.ACE inhibition or AT₁ antagonism is assessed by quantifying the %inhibition of pressor response to AngI or AngII, respectively. NEPinhibition is assessed by quantifying the potentiation of ANP-inducedelevation in urinary cGMP output.

Assay 4 In Vivo Evaluation of Antihypertensive Effects in the ConsciousSHR Model of Hypertension

Spontaneously hypertensive rats (SHR, 14-20 weeks of age) are allowed aminimum of 48 hours acclimation upon arrival at the testing site. Sevendays prior to testing, the animals are either placed on a restrictedlow-salt diet with food containing 0.1% of sodium for sodium depletedSHRs (SD-SHR) or are placed on a normal diet for sodium repleted SHRs(SR-SHR). Two days prior to testing, the animals are surgicallyimplemented with catheters into a carotid artery and the jugular vein(PE50 polyethylene tubing) connected via a PE10 polyethylene tubing to aselected silicone tubing (size 0.020 ID×0.037 OD×0.008 wall) for bloodpressure measurement and test compound delivery, respectively. Theanimals are allowed to recover with appropriate post operative care. Onthe day of the experiment, the animals are placed in their cages and thecatheters are connected via a swivel to a calibrated pressuretransducer. After 1 hour of acclimation, a baseline measurement is takenover a period of at least five minutes. The animals are then dosed i.v.with vehicle or test compound in ascending cumulative doses every 60minutes followed by a 0.3 mL saline to clear the catheter after eachdose. Data is recorded continuously for the duration of the study usingNotocord software (Kalamazoo, Mich.) and stored as electronic digitalsignals. In some studies, the effects of a single intravenous or oral(gavage) dose are monitored for at least 6 hours after dosing.Parameters measured are blood pressure (systolic, diastolic and meanarterial pressure) and heart rate.

Assay 5 In Vivo Evaluation of Antihypertensive Effects in the ConsciousDOCA-Salt Rat Model of Hypertension

CD rats (male, adult, 200-300 grams, Charles River Laboratory, USA) areallowed a minimum of 48 hours acclimation upon arrival at the testingsite before they are placed on a high salt diet. One week after thestart of the high salt diet, a DOCA-salt pellet (100 mg, 21 days releasetime, Innovative Research of America, Sarasota, Fla.) is implantedsubcutaneously and unilateral nephrectomy is performed. On 16 or 17 dayspost DOCA-salt pellet implantation, animals are implanted surgicallywith catheters into a carotid artery and the jugular vein with a PE50polyethylene tubing, which in turn was connected via a PE10 polyethylenetubing to a selected silicone tubing (size 0.020 ID×0.037 OD×0.008 wall)for blood pressure measurement and test compound delivery, respectively.The animals are allowed to recover with appropriate post operative care.

On the day of the experiment, each animal is kept in its cage andconnected via a swivel to a calibrated pressure transducer. After 1 hourof acclimation, a baseline measurement is taken over a period of atleast five minutes. The animals are then dosed i.v. with a vehicle ortest compound in escalating cumulative doses every 60 minutes followedby 0.3 mL of saline to flush the catheter after each dose. In somestudies, the effects of a single intravenous or oral (gavage) dose istested and monitored for at least 6 hours after dosing. Data is recordedcontinuously for the duration of the study using Notocord software(Kalamazoo, Mich.) and stored as electronic digital signals. Parametersmeasured are blood pressure (systolic, diastolic and mean arterialpressure) and heart rate. For cumulative and single dosing, thepercentage change in mean arterial pressure (MAP, mmHg) or heart rate(HR, bpm) is determined as described for Assay 4.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statues and regulations, all publications, patents andpatent applications cited herein are hereby incorporated by reference intheir entirety to the same extent as if each document had beenindividually incorporated by reference herein.

What is claimed is:
 1. A compound of formula I:

where: Z is:

where the amide portion of the compound of formula I is on the carbonatom between the oxygen ring atom and the nitrogen ring atom and the R³moiety is on the carbon ring atom adjacent to the oxygen ring atom; Aris selected from:

 R¹ is selected from —COOR, —SO₂NHC(O)R^(1a), tetrazolyl,

where R^(1a) is —C₁₋₆alkyl, —C₀₋₆alkylene-OR, —C₃₋₇cycloalkyl,—C₀₋₅alkylene-NRR, pyridyl, isoxazolyl, methylisoxazolyl, pyrrolidinyl,morpholinyl, and phenyl optionally substituted with halo; where each Ris independently selected from H and —C₁₋₆alkyl; a is 0, 1, or 2; R² isF; R³ is selected from —C₂₋₅alkyl and —O—C₁₋₅alkyl; R⁴ is—CH(R^(4b))COOR^(4c); where R^(4b) is H or —OH; and R^(4c) is H or—C₁₋₆alkyl; and R⁵ is selected from —C₁₋₆alkyl, —CH₂-furanyl,—CH₂-thiophenyl, benzyl, and benzyl substituted with one or more halo,—CH₃, or —CF₃ groups; where each ring in Ar is optionally substitutedwith 1 to 3 substituents independently selected from —OH, —C₁₋₆alkyl,—C₂₋₄alkenyl, —C₂₋₄alkynyl, —CN, halo, —O—C₁₋₆alkyl, —S—C₁₋₆alkyl,—S(O)—C₁₋₆alkyl, —S(O)₂—C₁₋₄alkyl, -phenyl, —NO₂, —NH₂, —NH—C₁₋₆alkyland —N(C₁₋₆alkyl)₂, wherein each alkyl, alkenyl and alkynyl isoptionally substituted with 1 to 5 fluoro atoms; or a pharmaceuticallyacceptable salt thereof.
 2. The compound of claim 1, where Ar is:


3. The compound of claim 1, where R¹ is —SO₂NHC(O)CH₃, —SO₂NHC(O)CH₂CH₃,—SO₂NHC(O)OCH₃, —SO₂NHC(O)OCH₂CH₃, —SO₂NHC(O)CH₂OCH₃, —SO₂NHC(O)CH₂OH,—SO₂NHC(O)CH(CH₃)OH, —SO₂NHC(O)C(CH₃)₂OH, —SO₂NHC(O)CH₂OCH₃,—SO₂NHC(O)(CH₂)₂OCH₃, —SO₂NHC(O)-cyclopropyl, —SO₂NHC(O)NH(CH₃),—SO₂NHC(O)N(CH₃)₂, —SO₂NHC(O)NH(CH₂CH₃), —SO₂NHC(O)C(CH₃)₂NH₂,—SO₂NHC(O)-2-pyridyl, —SO₂NHC(O)-4-pyridyl, —SO₂NHC(O)-5-isoxazolyl,—SO₂NHC(O)-3-isoxazolyl-5-methyl, —SO₂NHC(O)-1-pyrrolidyl,—SO₂NHC(O)-4-morpholinyl, —SO₂NHC(O)phenyl, —SO₂NHC(O)-2-fluorophenyl,1H-tetrazol-5-yl,


4. The compound of claim 1, where R³ is propyl, ethyl, butyl, or ethoxy.5. The compound of claim 1, where R⁴ is —CH(OH)COOH, or —CH₂COOH.
 6. Thecompound of claim 1, where R⁴ is —CH(OH)COOCH₃, or —CH₂COOCH₃.
 7. Thecompound of claim 1, where R⁵ is i-butyl, —CH₂-furan-2-yl,—CH₂-thiophen-3-yl, benzyl, 2-bromobenzyl, 2-chlorobenzyl,2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl, 2-methylbenzyl, or2-trifluoromethylbenzyl.
 8. A pharmaceutical composition comprising thecompound of claim 1 and a pharmaceutically acceptable carrier.
 9. Thepharmaceutical composition of claim 8 further comprising a secondtherapeutic agent selected from the group consisting of diuretics, β₁adrenergic receptor blockers, calcium channel blockers,angiotensin-converting enzyme inhibitors, AT₁ receptor antagonists,neprilysin inhibitors, non-steroidal anti-inflammatory agents,prostaglandins, anti-lipid agents, anti-diabetic agents, anti-thromboticagents, renin inhibitors, endothelin receptor antagonists, endothelinconverting enzyme inhibitors, aldosterone antagonists,angiotensin-converting enzyme/neprilysin inhibitors, vasopressinreceptor antagonists, and combinations thereof.
 10. The compound ofclaim 1, wherein R¹ is 1H-tetrazol-5-yl.
 11. The compound of claim 1,where R³ is —CH₂CH₂CH₃.
 12. The compound of claim 1, wherein R^(4b) is—OH.
 13. The compound of claim 1, wherein R^(4c) is H.
 14. The compoundof claim 1, wherein R⁵ is benzyl.
 15. A process for preparing thecompound of claim 1, comprising: (a) coupling a compound of formula 1with a compound of formula 2:

to produce a compound having the formula:

where: Ar* represents Ar—R^(1*), where R^(1*) is R¹ or a protected formof R¹; and R^(4*) represents R⁴ or a protected form of R⁴; andoptionally deprotecting the product when R^(1*) is a protected form ofR¹ and/or R^(4*) is a protected form of R⁴; (b) when R^(1*) is aprotected form of R¹ and/or R^(4*) is a protected form of R⁴,deprotecting the product of step (a) to produce a compound of formula I.16. An intermediate useful in the synthesis of the compound of claim 1,selected from the group comprising:

where: Ar* is Ar—R^(1*); R^(1*) is —SO₂NH—P⁶ or tetrazolyl-P⁴; R^(4*) is—CH(R^(4b))COO—P²; P² is a carboxy-protecting group; P⁴ is atetrazole-protecting group; and P⁶ is a sulfonamide-protecting group; ora salt thereof; wherein Z, Ar, R³, R⁴, R^(4b) and R⁵ are defined as inclaim 1.